Pieces Of DNA Research Articles

Design Principles of DNA-Barcodes for Nanopore-FET Readout, Based on Molecular Dynamics and TCAD Simulations.

Nanopore field-effect transistor (NP-FET) devices hold great promise as sensitive single-molecule sensors, which provide CMOS-based on-chip readout and are also highly amenable to parallelization. A plethora of applications will therefore benefit from NP-FET technology, such as large-scale molecular analysis (e.g., proteomics). Due to its potential for parallelization, the NP-FET looks particularly well-suited for the high-throughput readout of DNA-based barcodes. However, to date, no study exists that unravels the bit-rate capabilities of NP-FET devices. In this paper, we design DNA-based barcodes by labeling a piece of double-stranded DNA with dumbbell-like DNA structures. We explore the impact of both the size of the dumbbells and their spacing on achievable bit-rates. The conformational fluctuations of this DNA-origami, as observed by molecular dynamics (MD) simulation, are accounted for when selecting label sizes. An experimentally informed 3D continuum nanofluidic-nanoelectronic device model subsequently predicts both the ionic current and FET signals. We present a barcode design for a conceptually generic NP-FET, with a 14 nm diameter pore, operating in conditions corresponding to experiments. By adjusting the spacing between the labels to half the length of the pore, we show that a bit-rate of 78 kbit·s-1 is achievable. This lies well beyond the state-of-the-art of ≈40 kbit·s-1, with significant headroom for further optimizations. We also highlight the advantages of NP-FET readout based on the larger signal size and sinusoidal signal shape.

Site-specific immobilization of Cysteinyl leukotriene receptor 1 through enzymatic DNA-protein conjugation strategy for drug lead screening

Immobilization of functional protein, especially G protein-coupled receptors (GPCRs), is particularly significant in various fields such as the development of assays for diagnosis, lead compound screening, as well as drug-protein interaction analysis. However, there are still some challenges with the immobilized proteins such as undefined loads, orientations, and the loss of activity. Herein, we introduced a DNA conjugation strategy into the immobilization of Cysteinyl leukotriene receptor 1(CysLTR1) which enables exquisite molecular control and higher activity of the receptor. We used the bacterial relaxases VirD2 as an immobilized tag fused at the C terminus of CysLTR1. Tyrosine residue(Y29) at the core binding site of the VirD2 tag can react with the single-strand piece of DNA(T-DNA) in the form of a covalent bond. Inspired by this strategy, we developed a new immobilization method by mixing the T-DNA-modified silica gel with the cell lysate containing the expressed VirD2-tagged CysLTR1 for 1 hour. We found that the successful formation of DNA-protein conjugate enables the immobilization of CysLTR1 fast, site-specific, and with minimal loss of activity. The feasibility of the immobilized CysLTR1 was evaluated in drug-protein binding interaction by frontal analysis and adsorption energy distribution analysis. The binding of pranlukast, zafirlukast, and MK571 to the immobilized CysLTR1 was realized, and the association constants presented good agreement between the two methods. Rosmarinic acid was retained in the immobilized CysLTR1 column, and the in-vitro test revealed that the compound binds to the receptor in one type of binding site mode. Despite these results, we concluded that the DNA-protein conjugate strategy will probably open up the possibilities for capturing other functional proteins in covalent and site-specific modes from the complex matrices and the immobilized receptor preserves the potential in fishing out lead compounds from natural products.

Mass spectrometry study on SARS-CoV-2 recombinant vaccine with comprehensive separation techniques to characterize complex heterogeneity

SARS-CoV-2, the causative agent of COVID-19, has imposed a major public health threat, which needs effective therapeutics and vaccination strategies. Several potential candidate vaccines being rapidly developed are in clinical evaluation and recombinant vaccine has gained much attention thanks to its potential for greater response predictability, improved efficacy, rapid development and reduced side effects. Recombinant vaccines are designed and manufactured using bacterial, yeast cells or mammalian cells. A small piece of DNA is taken from the virus or bacterium against which we want to protect and inserted into the manufacturing cells. Due to the extremely complex heterogeneity of SARS-CoV-2 recombinant vaccine, single technology platform cannot achieve thorough and accurate characterization of such difficult proteins so integrating comprehensive technologies is essential. This study illustrates an innovative workflow employing multiple separation techniques tandem high-resolution mass spectrometry for comprehensive and in-depth characterization of SARS-CoV-2 recombinant vaccine, including ultra-high performance liquid chromatography (UHPLC), ion exchange chromatography (IEX) and imaged capillary isoelectric focusing (icIEF). The integrated methodology focuses on the importance of cutting-edge icIEF-MS online coupling and icIEF fractionation applied to revealing the heterogeneity secret of SARS-CoV-2 recombinant vaccine.

All who wander are not lost: the search for homology during homologous recombination.

Homologous recombination (HR) is a template-based DNA double-strand break repair pathway that functions to maintain genomic integrity. A vital component of the HR reaction is the identification of template DNA to be used during repair. This occurs through a mechanism known as the homology search. The homology search occurs in two steps: a collision step in which two pieces of DNA are forced to collide and a selection step that results in homologous pairing between matching DNA sequences. Selection of a homologous template is facilitated by recombinases of the RecA/Rad51 family of proteins in cooperation with helicases, translocases, and topoisomerases that determine the overall fidelity of the match. This menagerie of molecular machines acts to regulate critical intermediates during the homology search. These intermediates include recombinase filaments that probe for short stretches of homology and early strand invasion intermediates in the form of displacement loops (D-loops) that stabilize paired DNA. Here, we will discuss recent advances in understanding how these specific intermediates are regulated on the molecular level during the HR reaction. We will also discuss how the stability of these intermediates influences the ultimate outcomes of the HR reaction. Finally, we will discuss recent physiological models developed to explain how the homology search protects the genome.

Intron Retention: What that means for the biologist

Introns are non-coding segments within eukaryotic genes that are transcribed along with exons. They are subsequently removed during splicing, and their presence allows for generating diverse mRNA isoforms through the process called alternative splicing. The functional significance of introns going beyond mere "junk DNA" is questionable until recently; scientists identified that these pieces of junk DNA could hang around with the functional mRNA escaping splicing.  Intron retention is a type of alternative splicing, a process that occurs during the maturation of messenger RNA (mRNA) in eukaryotic cells. In eukaryotes, genes typically consist of exons (coding regions) and introns (non-coding regions). After transcription, the precursor mRNA (pre-mRNA) undergoes splicing, where introns are removed, and exons are joined together to form the mature mRNA. Intron retention, however, deviates from this typical splicing process. Instead of being entirely removed from the pre-mRNA, the mature mRNA may retain one or more introns. This results in the inclusion of intronic sequences within the final mRNA transcript. The retained introns may contain premature stop codons or regulatory elements that can influence gene expression and protein function. Intron retention is a form of alternative splicing because it generates multiple mRNA isoforms from a single gene. This diversity in mRNA transcripts can produce different protein isoforms with distinct functions or regulatory properties. The regulation of intron retention is complex and involves various cellular factors, including splicing machinery components and RNA-binding proteins. Research on intron retention has revealed its importance in various biological processes, including development, cell growth and  differentiation, and diseases like cancer. Aberrant intron retention has been associated with certain genetic disorders and cancers. Understanding the mechanisms and functional consequences of intron retention is crucial for unraveling the complexity of gene regulation and its impact on cellular processes. With an increasing output of data from high throughput assays line RNA Seq, scRNA-Seq, etc., it's high time data scientists look into these signatures in the context of disease pathology. This may well be the tip of the iceberg.    

Abstract IA017: Extrachromosomal DNA (ecDNA): Cancer’s dynamic circular genome

Abstract Human genes are arranged on 23 pairs of chromosomes, but in cancer, tumor-promoting genes and regulatory elements can free themselves from chromosomes and relocate to circular, extrachromosomal pieces of DNA (ecDNA). ecDNA poses one of the greatest challenges for cancer patients, affecting children and adults and women and men, with a wide variety of cancer types. Although ecDNA was first observed more than 50 years ago, its critical importance has only recently come to light. ecDNA, because of its non-chromosomal inheritance, drives high-copy number oncogene amplification and enables tumors to evolve their genomes rapidly, contributing to treatment resistance and shorter survival for patients. Furthermore, the circular ecDNA architecture fundamentally alters gene regulation and transcription. In this talk, I will discuss recent collaborative discoveries that highlight how the higher order, spatial organization of ecDNA, and its mechanism of inheritance, contributes to aggressive tumorigenesis, accelerated evolution, and treatment resistance. Citation Format: Paul Mischel. Extrachromosomal DNA (ecDNA): Cancer’s dynamic circular genome [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr IA017.

Applying Lessons Learned from Developing Exon Skipping for Duchenne to Developing Individualized Exon Skipping Therapy for Patients with Neurodegenerative Diseases

AbstractAntisense oligonucleotides (ASOs) are short, modified pieces of DNA that are chemically modified. They can be used to induce exon skipping and treat Duchenne muscular dystrophy (DMD) patients by interfering with the splicing process so mutated dystrophin transcripts become readable allowing production of partially functional dystrophin proteins, rather than nonfunctional dystrophins. After over 2 decades of research, 4 ASOs are FDA approved for DMD, but clinical effects are suboptimal due to limited delivery to skeletal muscle. At the same time, ASOs for brain diseases result in much more functional impact, because local delivery allows higher exposure to the target tissue at a low dose and infrequent treatment regimen. This has opened the way to develop ASOs in an individualized setting, as was exemplified by the development of Milasen to treat a patient with CLN7 Batten disease.In this perspective paper I will share my personal journey as one of the pioneers of ASO-mediated exon skipping development for DMD, currently applying expertise gained and lessons learned along the way to develop exon skipping ASOs for eligible patients with genetic brain diseases in a national and international setting.1 Duchenne and Antisense-Mediated Exon Skipping2 Opportunities for Treating Central Nervous System Diseases and Developing Individualized ASOs for Central Nervous System Diseases3 Collaborative Spirit to Develop Individualized Treatments Globally4 Global Implementation5 Concluding Remarks

CRISPR- Cas9 Technology: Mechanism and Its Application in The Field of Entomology

The field of life science research has undergone a revolution thanks to the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated protein (CRISPR/Cas) system, which provides a multitude of opportunities for modifying, identifying, visualising, and annotating particular DNA or RNA sequences in diverse organisms. In this technique, foreign DNA pieces, known as spacers, are inserted into CRISPR cassettes. These spacers are then transcribed into CRISPR arrays and processed to produce guide RNA (gRNA). The Cas proteins that the CRISPR arrays encode serve as the enzymatic machinery required to obtain new spacers that specifically target invasive genetic elements. Several Cas proteins, such as Cas9, Cas12, Cas13, and Cas14, have been used to create novel tools for genome engineering due to their programmable sequence specificity. The ability to manipulate and edit nucleic acid sequences in living cells from a wide variety of organisms has been made possible by these Cas variants, which have greatly advanced genetic research and the CRISPR/Cas tool. The CRISPR Cas-9 technology has applications in many areas of entomology, including the genetics of honeybees and plants that produce insecticidal compounds. CRISPR/Cas9 technology has transformed entomology by providing precise tools for gene editing and genetic manipulation in insects. This has enabled advancements in fundamental research, disease vector control, and pest management, with the potential to reduce the environmental and economic impact of insect pests in agriculture and public health.

Topological structure of complex predictions

Current complex prediction models are the result of fitting deep neural networks, graph convolutional networks or transducers to a set of training data. A key challenge with these models is that they are highly parameterized, which makes describing and interpreting the prediction strategies difficult. We use topological data analysis to transform these complex prediction models into a simplified topological view of the prediction landscape. The result is a map of the predictions that enables inspection of the model results with more specificity than dimensionality-reduction methods such as tSNE and UMAP. The methods scale up to large datasets across different domains. We present a case study of a transformer-based model previously designed to predict expression levels of a piece of DNA in thousands of genomic tracks. When the model is used to study mutations in the BRCA1 gene, our topological analysis shows that it is sensitive to the location of a mutation and the exon structure of BRCA1 in ways that cannot be found with tools based on dimensionality reduction. Moreover, the topological framework offers multiple ways to inspect results, including an error estimate that is more accurate than model uncertainty. Further studies show how these ideas produce useful results in graph-based learning and image classification.

Standardized Representation of Parts and Assembly for Build Planning.

The design and construction of genetic systems, in silico, in vitro, or in vivo, often involve the handling of various pieces of DNA that exist in different forms across an assembly process: as a standalone "part" sequence, as an insert into a carrier vector, as a digested fragment, etc. Communication about these different forms of a part and their relationships is often confusing, however, because of a lack of standardized terms. Here, we present a systematic terminology and an associated set of practices for representing genetic parts at various stages of design, synthesis, and assembly. These practices are intended to represent any of the wide array of approaches based on embedding parts in carrier vectors, such as BioBricks or Type IIS methods (e.g., GoldenGate, MoClo, GoldenBraid, and PhytoBricks), and have been successfully used as a basis for cross-institutional coordination and software tooling in the iGEM Engineering Committee.

Unusual modifications of protein biomarkers expressed by plasmid, prophage, and bacterial host of pathogenic Escherichia coli identified using top‐down proteomic analysis

RationalePathogenic bacteria often carry prophage (bacterial viruses) and plasmids (small circular pieces of DNA) that may harbor toxin, antibacterial, and antibiotic resistance genes. Proteomic characterization of pathogenic bacteria should include the identification of host proteins and proteins produced by prophage and plasmid genomes.MethodsProtein biomarkers of two strains of Shiga toxin–producing Escherichia coli (STEC) were identified using antibiotic induction, matrix‐assisted laser desorption/ionization tandem time‐of‐flight (MALDI‐TOF‐TOF) tandem mass spectrometry (MS/MS) with post‐source decay (PSD), top‐down proteomic (TDP) analysis, and plasmid sequencing. Alphafold2 was also used to compare predicted in silico structures of the identified proteins to prominent fragment ions generated using MS/MS‐PSD. Strain samples were also analyzed with and without chemical reduction treatment to detect the attachment of pendant groups bound by thioester or disulfide bonds.ResultsShiga toxin was detected and/or identified in both STEC strains. For the first time, we also identified the osmotically inducible protein (OsmY) whose sequence unexpectedly had two forms: a full and a truncated sequence. The truncated OsmY terminates in the middle of an α‐helix as determined by Alphafold2. A plasmid‐encoded colicin immunity protein was also identified with and without attachment of an unidentified cysteine‐bound pendant group (~307 Da). Plasmid sequencing confirmed top‐down analysis and the identification of a promoter upstream of the immunity gene that is activated by antibiotic induction, that is, SOS box.ConclusionsTDP analysis, coupled with other techniques (e.g., antibiotic induction, chemical reduction, plasmid sequencing, and in silico protein modeling), is a powerful tool to identify proteins (and their modifications), including prophage‐ and plasmid‐encoded proteins, produced by pathogenic microorganisms.

Altering under-represented DNA sequences elevates bacterial transformation efficiency.

A cornerstone of bacterial molecular biology is the ability to genetically manipulate the microbe under study. Many bacteria are difficult to manipulate genetically, a phenotype due in part to robust removal of newly acquired DNA, for example, by restriction-modification (R-M) systems. Here, we report approaches that dramatically improve bacterial transformation efficiency, piloted using a microbe that is challenging to transform due to expression of many R-M systems, Helicobacter pylori. Initially, we identified conditions that dampened expression of several R-M systems and concomitantly enhanced transformation efficiency. We then identified an approach that would broadly protect newly acquired DNA. We computationally predicted under-represented short DNA sequences in the H. pylori genome, with the idea that these sequences reflect targets of sequence-based surveillance such as R-M systems. We then used this information to modify and eliminate such sites in antibiotic resistance cassettes, creating a "stealth" version. Modifying antibiotic resistance cassettes in this way resulted in significantly higher transformation efficiency compared to non-modified cassettes, a response that was genomic loci independent. Our results suggest that avoiding R-M systems, via modification of under-represented DNA sequences or transformation conditions, is a powerful method to enhance DNA transformation. Our approach to identify under-represented sequences is applicable to any microbe with a sequenced genome.IMPORTANCEManipulating the genomes of bacteria is critical to many fields. Such manipulations are made by genetic engineering, which often requires new pieces of DNA to be added to the genome. Bacteria have robust systems for identifying and degrading new DNA, some of which rely on restriction enzymes. These enzymes cut DNA at specific sequences. We identified a set of DNA sequences that are missing normally from a bacterium's genome, more than would be expected by chance. Eliminating these sequences from a new piece of DNA allowed it to be incorporated into the bacterial genome at a higher frequency than new DNA containing the sequences. Removing such sequences appears to allow the new DNA to fly under the bacterial radar in "stealth" mode. This transformation improvement approach is straightforward to apply and likely broadly applicable.

B-291 Proximity Labeling at Sites of Protein-Protein Interactions Using a Switchable DNA Catalyst

Abstract Background Extracellular protein-protein interactions (PPIs) play a crucial role in cell-cell adhesion, communication, and signaling. The dysregulation of PPIs in cancer caused by differential protein expression can drive oncogenic factors. Traditional methods to study PPIs disfavor weak or transient interacting protein partners, leaving them undiscovered. Proximity labeling (PL) is an emerging tool in which a catalyst is attached to a protein of interest (POI) and creates reactive species that tag nearby endogenous proteins. Tagged proteins are subsequently isolated and identified as potential interacting partners with the POI. If PL could be activated at a more specific subcellular location, such as the site of a PPI, more detailed information could be unveiled concerning the interactome of cancer-relevant PPIs. Recent advances in PL involve the use of a synthetic catalyst that can be localized to a POI via an antibody or ligand and avoids the need for PL enzyme-POI fusion proteins to be constructed, allowing for analysis of primary tissue samples. Methods To create a PL catalyst with pseudo-allosteric regulation, we created a switchable DNA catalyst. The DNA catalyst is composed of synthetic photocatalyst and quencher moieties covalently linked to DNA oligomers such that when hybridized together the photocatalyst is quenched and inactive. The DNA scaffold affords conformational control that is utilized to change the distance between photocatalyst and quencher, thus regulating photocatalyst activity. Split triggers composed of DNA oligos that bind to catalyst-DNA and simultaneously disrupt quencher-DNA hybridization were used to create an AND logic gate where both split triggers need to be in proximity to activate the PL catalyst. The split triggers were localized to POIs via connection to aptamers, short pieces of synthetic DNA that bind targets. Using the c-Met homodimer as a model system, we first labeled c-Met expressing HEK293T cells with aptamer-split trigger DNA oligomers as well as the quenched photocatalyst. After the catalyst was activated at the site of the PPI, we performed proximity labeling using a biotin-phenol substrate in the presence of an oxidizing agent and 450 nm light. Fluorescent streptavidin was used to visualize sites of biotinylation and detected using fluorescence microscopy. Results We observed extracellular biotin signal only of c-Met transfected cells as evidenced by the expression of a co-transfection plasmid encoding a fluorescent nuclear protein. When one of the split triggers had the aptamer motif removed, meaning it couldn’t be localized to the cell surface, no labeling was observed which shows that both split probes must be localized to the cell surface to activate labeling. Additionally, controls including omission of light, photocatalyst, or biotin phenol showed no labeling. Using a free-floating version of the photocatalyst to label cells, we confirmed that lysates contained biotinylated proteins via western blot. Conclusion We localized two split triggers to interacting proteins to activate a PL catalyst and performed proximity labeling at the site of the PPI. This technique demonstrates non-genetic split proximity labeling to enable downstream analysis of cancer cell proteomes in an ultra-specific manner and expands the toolkit for spatiotemporally controlled proximity labeling.

A CRISPR-based rapid DNA repositioning strategy and the early intranuclear life of HSV-1.

The relative positions of viral DNA genomes to the host intranuclear environment play critical roles in determining virus fate. Recent advances in the application of chromosome conformation capture-based sequencing analysis (3 C technologies) have revealed valuable aspects of the spatiotemporal interplay of viral genomes with host chromosomes. However, to elucidate the causal relationship between the subnuclear localization of viral genomes and the pathogenic outcome of an infection, manipulative tools are needed. Rapid repositioning of viral DNAs to specific subnuclear compartments amid infection is a powerful approach to synchronize and interrogate this dynamically changing process in space and time. Herein, we report an inducible CRISPR-based two-component platform that relocates extrachromosomal DNA pieces (5 kb to 170 kb) to the nuclear periphery in minutes (CRISPR-nuPin). Based on this strategy, investigations of herpes simplex virus 1 (HSV-1), a prototypical member of the human herpesvirus family, revealed unprecedently reported insights into the early intranuclear life of the pathogen: (I) Viral genomes tethered to the nuclear periphery upon entry, compared with those freely infecting the nucleus, were wrapped around histones with increased suppressive modifications and subjected to stronger transcriptional silencing and prominent growth inhibition. (II) Relocating HSV-1 genomes at 1 hr post infection significantly promoted the transcription of viral genes, termed an 'Escaping' effect. (III) Early accumulation of ICP0 was a sufficient but not necessary condition for 'Escaping'. (IV) Subnuclear localization was only critical during early infection. Importantly, the CRISPR-nuPin tactic, in principle, is applicable to many other DNA viruses.

Rapid detection of myeloid neoplasm fusions using single-molecule long-read sequencing.

Recurrent gene fusions are common drivers of disease pathophysiology in leukemias. Identifying these structural variants helps stratify disease by risk and assists with therapy choice. Precise molecular diagnosis in low-and-middle-income countries (LMIC) is challenging given the complexity of assays, trained technical support, and the availability of reliable electricity. Current fusion detection methods require a long turnaround time (7-10 days) or advance knowledge of the genes involved in the fusions. Recent technology developments have made sequencing possible without a sophisticated molecular laboratory, potentially making molecular diagnosis accessible to remote areas and low-income settings. We describe a long-read sequencing DNA assay designed with CRISPR guides to select and enrich for recurrent leukemia fusion genes, that does not need a priori knowledge of the abnormality present. By applying rapid sequencing technology based on nanopores, we sequenced long pieces of genomic DNA and successfully detected fusion genes in cell lines and primary specimens (e.g., BCR::ABL1, PML::RARA, CBFB::MYH11, KMT2A::AFF1) using cloud-based bioinformatics workflows with novel custom fusion finder software. We detected fusion genes in 100% of cell lines with the expected breakpoints and confirmed the presence or absence of a recurrent fusion gene in 12 of 14 patient cases. With our optimized assay and cloud-based bioinformatics workflow, these assays and analyses could be performed in under 8 hours. The platform's portability, potential for adaptation to lower-cost devices, and integrated cloud analysis make this assay a candidate to be placed in settings like LMIC to bridge the need of bedside rapid molecular diagnostics.

Multisite transformation in Neisseria gonorrhoeae: insights on transformations mechanisms and new genetic modification protocols.

Natural transformation, or the uptake of naked DNA from the external milieu by bacteria, holds a unique place in the history of biology. This is both the beginning of the realization of the correct chemical nature of genes and the first technical step to the molecular biology revolution that sees us today able to modify genomes almost at will. Yet the mechanistic understanding of bacterial transformation still presents many blind spots and many bacterial systems lag behind power horse model systems like Escherichia coli in terms of ease of genetic modification. Using Neisseria gonorrhoeae as a model system and using transformation with multiple DNA molecules, we tackle in this paper both some aspects of the mechanistic nature of bacterial transformation and the presentation of new molecular biology techniques for this organism. We show that similarly to what has been demonstrated in other naturally competent bacteria, Neisseria gonorrhoeae can incorporate, at the same time, different DNA molecules modifying DNA at different loci within its genome. In particular, co-transformation of a DNA molecule bearing an antibiotic selection cassette and another non-selected DNA piece can lead to the integration of both molecules in the genome while selecting only through the selective cassette at percentages above 70%. We also show that successive selections with two selection markers at the same genetic locus can drastically reduce the number of genetic markers needed to do multisite genetic modifications in Neisseria gonorrhoeae. Despite public health interest heightened with the recent rise in antibiotic resistance, the causative agent of gonorrhea still does not possess a plethora of molecular techniques. This paper will extend the techniques available to the Neisseria community while providing some insights into the mechanisms behind bacterial transformation in Neisseria gonorrhoeae. We are providing a suite of new techniques to quickly obtain modifications of genes and genomes in the Neisserial naturally competent bacteria.

A graph neural network-based interpretable framework reveals a novel DNA fragility–associated chromatin structural unit

BackgroundDNA double-strand breaks (DSBs) are among the most deleterious DNA lesions, and they can cause cancer if improperly repaired. Recent chromosome conformation capture techniques, such as Hi-C, have enabled the identification of relationships between the 3D chromatin structure and DSBs, but little is known about how to explain these relationships, especially from global contact maps, or their contributions to DSB formation.ResultsHere, we propose a framework that integrates graph neural network (GNN) to unravel the relationship between 3D chromatin structure and DSBs using an advanced interpretable technique GNNExplainer. We identify a new chromatin structural unit named the DNA fragility–associated chromatin interaction network (FaCIN). FaCIN is a bottleneck-like structure, and it helps to reveal a universal form of how the fragility of a piece of DNA might be affected by the whole genome through chromatin interactions. Moreover, we demonstrate that neck interactions in FaCIN can serve as chromatin structural determinants of DSB formation.ConclusionsOur study provides a more systematic and refined view enabling a better understanding of the mechanisms of DSB formation under the context of the 3D genome.

The Long and Winding Road: On-Demand DNA Synthesis in High Demand

GEN BiotechnologyVol. 2, No. 2 News FeatureFree AccessThe Long and Winding Road: On-Demand DNA Synthesis in High DemandJonathan D. GrinsteinJonathan D. GrinsteinE-mail Address: jgrinstein@liebertpub.comSenior Editor, GEN Media Group.Search for more papers by this authorPublished Online:18 Apr 2023https://doi.org/10.1089/genbio.2023.29087.jdgAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Accessibility to DNA writing technology is growing thanks to the commercialization of these techniques through services ranging from benchtop DNA printers to custom synthesis.The state of DNA synthesis is a bit like humanity's journey to the moon—just because something has been achieved before doesn't mean we should cease figuring out how to do it better.”That is how Harold P. de Vladar, CEO and founder of long-synthetic DNA service provider Ribbon Biolabs, sees the vast opportunity in the DNA synthesis space. de Vladar says that the DNA synthesis equivalent to the Apollo 11 moon landing was Craig Venter's landmark work on assembling the 5386 bp bacteriophage ΦX174 from short single strands of synthetic, commercially available DNA known as oligonucleotides.1 Venter and his team at the J. Craig Venter Institute built the ΦX174 genome using an adaptation of polymerase chain reaction (PCR) called polymerase cycle assembly2 in 2002–2003—just after the sequencing of the human genome in the early 2000s.This problem still existed until a few years ago. “When I was still a researcher, I confronted the problem of making long DNA by wanting to synthesize a library of small phage genomes, around 4000 bp, and that was an impossible project. I wanted like 250 sequences, and this was unachievable,” de Vladar said.About 10 years ago, de Vladar saw that a company called Twist Bioscience was starting to appear in the news. He came to the realization that people were still interested in synthesizing long DNA. “Superficially, synthesizing DNA seemed solved, but when you start looking into the details, how the cost per base per has dropped for sequencing and so on, you realize that this was far from solved” (Box 1).Box 1. Learning to Read Before Learning to WriteThe cost of sequencing has plummeted for the past 10–15 years at a pace that has outstripped Moore's law. The cost of a human genome sequence decreased from an estimated $1 million in 2007, to $1000 in 2014, and today it is nearing $100, with the Illumina NovaSeq X series and Ultima Genomics UG100 claiming costs of $200 and $100 per genome, respectively. But the affordability of custom DNA synthesis has not kept pace and the cost remains relatively high. For the past several years, there has been a general trend toward gene synthesis costs falling to $0.01 per bp, as opposed to less than the $1–2 per Gigabase offered by the latest next-generation sequencing tools.7In 2018, de Vladar launched Ribbon Biolabs in Vienna, Austria. The DNA synthesis company can reliably construct 1-kb fragments in a day, which can be assembled into larger molecules of 10–20 kb. To do so, Ribbon algorithmically processes a sequence into smaller pieces that get sorted into a decision tree to identify the best combination of 10- and 12-mer oligos (from their biobank of nearly 80,000 oligos) for automated assembly. The true turnaround time, including sequencing-based verification and delivery, for the small company (with just a few dozen employees), is ∼3 days. The goal for de Vladar and Ribbon Biolabs is to scale the gene synthesis from the library approach to 1000 kb per day.Assembly Versus SynthesisBy improving enzymes and assembly standards, the accuracy and number of DNA molecules that can be combined in a single step have improved. In the past decade, a plethora of powerful new DNA assembly methods—including Gibson Assembly3—have been developed. These assembly methods have been applied in the construction of a minimal bacterial genome4 and synthetic yeast chromosomes.5Assembly approaches to DNA synthesis have several drawbacks: they can be compromised by impurities of synthetic oligonucleotides, the dependence of the method on sequence confirmation from an individual clone, and the reliance on high-fidelity proofreading PCR enzymes, which must be used to copy constructed genes to prevent mutations during amplification.Ribbon Biolabs' tiered approach is limited by the physical size of the synthesized molecules. “DNA that's 20–40 kb is a really complex molecule,” said de Vladar. “The reactions are not that efficient anymore. But that's not even the worst of the problem: longer DNA molecules break due to sheer force. The assembly itself isn't the issue; it's the manipulation of the DNA molecules. It's a problem we're also trying to solve, and we have some ideas in R&D. But it's not going to just print 30 kb at the press of a button.”Furthermore, not everyone agrees that assembly is DNA synthesis. “When we talk about DNA synthesis, we have to be a little cautious because some people talk about oligonucleotide synthesis and other people talk about gene assembly,” said Sylvain Gariel, cofounder and COO of DNA Script, a company that chemically synthesizes DNA.Since the structure of DNA was first solved 70 years ago, substantial milestones have been achieved, paving the way for the DNA synthesis industry. For the past 40 years, scientists have taken steady steps to figure out the chemistry behind the step-by-step creation of DNA, nucleotide by nucleotide. Chemical methods were developed to reliably provide short DNA chains, typically <200 nucleotides (oligonucleotides). These methods were made to work best with automatic synthesizers, which are now needed for gene engineering and sequencing.Next, scientists devised methods to create DNA that is longer and more complex than oligonucleotides by using enzymes with or without DNA templates. For instance, the basic technology for DNA Script is template-independent enzymatic oligonucleotide synthesis. Companies have turned these chemical synthesis methods into products and services, such as benchtop DNA printers and custom synthesis, which make DNA synthesis possible for people who are not experts.On March 9, 2023, Ansa Biotechnologies, Inc. reported that they had successfully made the world's longest DNA oligonucleotide in a single synthesis. The 1005-base sequence codes for a key part of an adeno-associated virus vector used to develop gene therapy. It has complex features, such as strong secondary structures and a high GC content, that make it very hard to make with traditional methods that require putting together shorter oligonucleotides.Do It Yourself DNA PrintingAs a metabolite engineer, Gariel was limited by the acquisition of DNA constructs. He had to wait weeks or even months to obtain long stretches of DNA for tens or even hundreds of genes. At DNA Script, Gariel is making strides to commercializing a benchtop DNA synthesis device called Syntax to bypass this bottleneck (Fig. 1).FIG. 1. DNA script's EDS benchtop platform.Powered by EDS Technology, the DNA Script's SYNTAX System is a DNA printer that enables on-demand synthesis of custom DNA oligos in a benchtop solution without the need for specialized infrastructure or technical skills. Oligos are enzymatically synthesized in a cyclic two-step process. The first synthesis step first is for elongation, in which the terminal deoxynucleotidyl transferase enzyme adds a single nucleotide to a predefined initiator DNA that is attached to a solid support that allows for the control of synthesis scale and manipulating the growing oligo strands throughout the EDS process. The second synthesis step is for deprotection, where the reversible terminator of the nucleotide is removed, leaving the strand ready to be elongated again. Steps 1 and 2 are repeated until the longest oligo on the plate is completed. EDS, enzymatic DNA synthesis.“Our end goal in the market was, if I can do it on my bench stuff, I'm suddenly cutting out all the times that the service provider requires,” Gariel explained. “I'm not giving control to a service provider as long as I have an instrument and the reagents that I need to run it. I can do it on the benchtop without any specific infrastructure requirements other than a molecular biology space and an electric plug.”The Syntax instrument is a benchtop DNA synthesizer similar in size to the HiSeq sequencer from Illumina. This synthesizer can generate 60-bp oligonucleotides in a pure form for immediate use within 6 h.To make DNA synthesis safer and more accessible, DNA Script has addressed the danger posed by phosphoramidite chemistry (Box 2), both to the user and the environment. Gariel notes that this is why this chemistry comes with strong constraints in terms of infrastructure and dedicated labor.Box 2. Phosphoramidite SynthesisAdvances in solid-phase synthesis—the synthesis of chemical compounds whereby the reactant molecule is chemically bound to an insoluble material and reagents are added in the solution phase—inspired the groundbreaking development of phosphoramidite chemistry for DNA synthesis in the 1980s by Marvin H. Caruthers of the University of Colorado Boulder.8 In the 1980s, the first automated DNA synthesizer resulted from a collaboration with Caruthers, and was based on Caruthers' work elucidating the chemistry of phosphoramidite oligonucleotide synthesis. Applied Biosystems used this method to make the first automated DNA synthesizer. This made it easier for people to get synthetic oligonucleotides. But the phosphoramidite method of making DNA has some problems, such as phosphoramidite that does not stay stable on the bench, the need to use a lot of organic solvents, and the inability to make poly-repeat sequences.Synthesizing, processing, and purifying oligonucleotides is a labor-intensive process that is still mostly done by service providers. Therefore, synthesis capabilities have become centralized within specialty reagent manufacturers. Leading companies such as Agilent Technologies, GenScript, Integrated DNA Technologies, ThermoFisher, TriLink, Dharmacon, Twist Bioscience, and others make custom DNA (and RNA) on demand in a variety of formats. There is a range of instruments for users who wish to decrease the lead time for such services, such as Cytiva's ÄKTA oligonucleotide synthesizers, which can be purchased and operated on a daily basis.In the past, molecular biology used short DNA sequences such as primers for polymerase chain reaction or probes for molecular detection, amplification, and change. Now, scientists are looking for longer sequences with different parts, such as whole genomes with accuracy down to a single base, which must be put together from scratch. The phosphoramidite method cannot work with such long sequences because it is less effective at making pure DNA after about 200-bp oligonucleotide sequences.Using a different method, longer sequences must be put together from smaller strands in stages where mistakes are fixed. In this way, enzymatic approaches are most appealing because they can be used on a large scale, are able to target specific groups, and are good for the environment. Enzymes can mediate mismatch recognition, enabling the selective annealing of complementary strands, reduce the number of steps in each elongation cycle by eliminating the need for coupling reagents, and decrease the dependence on organic solvents. Enzymes can promote synthesis with or without DNA templates, through amplification or the synthesis of de novo sequences.“Those chemicals are incredibly harsh—they're volatile and carcinogenic,” said Gariel. “Typically, you don't want to deal with them in the lab. We designed an enzymatic DNA synthesis (EDS) technology that is water-based, which becomes incredibly versatile and easy to automate.”Matthew Hayes, chief technology officer and cofounder of Evonetix, says that most attempts to make DNA have focused on shrinking the technology. Evonetix has taken a very different approach. “We've asked ourselves, ‘What's wrong with DNA synthesis?’ ‘Why is it fundamentally difficult to make long pieces of DNA, and can we come up with a solution that solves that from the ground up?’” Hayes explained.“There are people today who can make machines that will make a small number of high-yield oligos and PCR primers using column-based synthesis. But if you want to synthesize an array of low molecular quantity but a high diversity pool, you're pretty much restricted to service providers.” Evonetix plans to give users instruments that can synthesize a pool of oligos using semiconductor-based chips (Fig. 2). “We're also able to instruct our machine to assemble that pool into a much longer double stranded DNA template,” Hayes said.FIG. 2. Evonetix silicon chips for benchtop DNA synthesis.The proprietary semiconductor-based silicon chip developed by Evonetix actively controls DNA synthesis, gene assembly, and error correction at thousands of discrete sites across its surface. These chips are part of a synthesis cartridge that contains both the synthesis and assembly chips plus all the reagents required for a single synthesis run, enabling simple plug-and-play operation. The synthesis cartridge is then inserted into the benchtop instrument, which manages the electronic and fluidic connections needed to prepare the DNA sequences specified by the user through automation. Chip dimensions: 24 × 21 mm.Chemical DNA Synthesis as a ServiceThis is not the first time that benchtop DNA synthesizers have hit the market. Emily Leproust, CEO of Twist Bioscience, remembers when people stopped buying benchtop DNA synthesizers. “I started my PhD in DNA synthesis in 1996, and we bought one of the last decentralized desktop oligo synthesizers in 1998,” Leproust told GEN Biotechnology.“I'm from a generation that remembers when people stopped buying DNA synthesizers. The reason why they stopped is that it was just cheaper and faster to send it out. A lot of people have forgotten that we used to have the benchtop DNA synthesizer, and that market just died because the centralized approach is faster and cheaper.”Among the several thousand Twist customers, Leproust receives a few requests each year asking if users can get the chip and DNA synthesis machine in their laboratory, to which she would oblige. “There are maybe a few niche applications where you are willing to pay a lot more to have the synthesis done onsite,” said Leproust. “But if it's onsite, it's going to be a lot more expensive—you will never beat the price in a centralized facility.”Leproust says Twist has “unlimited scalability. When I had my desktop DNA synthesizer in graduate school, I could make eight oligos. Now, the desktops can do around 96. But what if you want to make 500 oligos? You have to run the instrument five times in a row. If you want 10,000 oligos, you have to run it 100 times in a row! Our range is super flexible.”DNA synthesis companies such as Twist offer the ability to make complex and large amounts of DNA quickly at prices that are finally becoming more affordable. Twist's silicon-based high-throughput gene synthesis platform makes high-quality gene fragments for as little as $0.07 per bp and perfect clonal gene sequences verified by Next-Generation Sequencing for as little as $0.09 per bp.“Our technology can make millions of oligos at the same time, up to 300 bases with a quality of about one error in 2–3000 bases, and we can assemble up to 5000 bases in a construct,” she said. “If you want fragments of 1 kb in length, we can do that for $70 and ship them to you in four days, and we can make more than 1 million of these 1000-base fragments a year.”Although Leproust thinks that Twist's technology can push past 300-base oligos, the synthesis of the oligos is relatively slow compared with stitching them together. “If we were to make 1,000 bases all by synthesis, it would not be faster than what we shipped today in four days and you'll lose out on speed, quality, and cost,” said Leproust. “There's some vanity in saying, ‘I can make a 1,000-bp fragment.’ But if it's more expensive, slower, and of lower quality, that's not what the customer wants. We're more into what moves the needle for the customer. I'm not into running science experiments. We are a high-throughput, high-scale, and low-cost operation with a very nice gross margin, and we have to deliver revenue growth quarterly.”Twist is working on several fronts to improve their DNA synthesis as a service. One dimension of Twist's R&D is for the silicon chip. Leproust said that they are trying to create higher density DNA synthesis. “We can make a million oligos on the current chip, which is the size of a big cell phone or a 96-well plate,” said Leproust (Fig. 3). “The new chip is 24 times smaller, about the size of a postage stamp, and we can make 256 million oligos on these. That's an improvement in amount and cost over 6,000 × . Our roadmap for miniaturization and to lower the cost is to go from 256 million to 3 billion, then 10 billion, then 50 billion oligos per chip.”FIG. 3. Twist's silicon platform for DNA synthesis as a service.Twist Bioscience developed a proprietary semiconductor-based synthetic DNA manufacturing process featuring a high-throughput silicon platform that enables the miniaturization of the chemistry necessary for DNA synthesis. Twist can “write” >1 million pieces of DNA on a single silicon chip the size of a typical cell phone or 96-well plate.The second dimension pertains to speed. It currently takes 4 days from ordering to receiving 1000 bp synthesized DNA on the FedEx truck. Leproust thinks Twist can probably cut that in half to 2 days. A third consideration is the length of the gene fragments. Twist's current maximum is about 1.8 kb, but Leproust wants to stretch that to 5 kb. The fourth aspect is the mass that the customer needs. Leproust says some customers want femtomoles, whereas others might need 100 kg. Twist is on the low side of this range but is working on extending the quantities that customers want.Hot Take: Protein WritingIn an increasingly crowded field, the best way to take the lead in DNA synthesis is to make things orders of magnitude better than the state of the art. That is why Michael Chen, CEO and founder of Nuclera, decided to pivot. “We didn't see a trajectory where enzymatic DNA synthesis would result in a 10 × or 100 × improvement in something for the customer.”Chen and his cofounders settled on a different question: What is the bottleneck in research today? The key bottleneck for all the companies in the EDS space, Chen argues, is actually making the proteins to write DNA.Trained as an X-ray crystallographer, “people often asked me what the bottleneck in the process was?” Chen said. “The natural assumption is that it's the crystallography, which took me 3–6 months to learn. But what took me years was just getting good protein in the first place to carry out my PhD project! That sounds a lot like drug discovery, where researchers are spending months, maybe even years, to get target proteins. Today's protein scientists are in the lab for weeks performing an incredibly labor-intensive process to grow, manipulate, break, and purify cells. It's a multi-billion-dollar industry, so it's a huge opportunity.”Chen saw an opportunity to take Nuclera's core digital microfluidics technology and collaborate in a strategic partnership with ePaper display creator E Ink. “The supply chain and research to make ePaper displays have a lot to do with the type of droplet automation technology that we're working on and commercializing at Nuclera. We've made [the eDrop lab-on-a-chip technology] to move thousands of droplets in a programmable fashion in what we call a ‘pipette and forget’ process. The researcher pipettes directly into the cartridge and then can walk away.”In the FoldWhether using assembly or chemical oligonucleotide synthesis methods, the generation of long DNA is a reality and has a growing list of biotechnological applications beyond making proteins and synthetic life. DNA synthesis can be used to make vaccines, gene therapies, DNA data storage, DNA origami (Box 3), nanobots,6 and plants that are resistant to climate change. This has led to a huge demand for mass production of long synthetic DNA and, with it, a surge in the development and commercialization of DNA synthesis techniques.Box 3. DNA OrigamiDNA origami consists of using short oligos (“staples”) to guide the folding of a long (“scaffold”) strand and enable the assembly of complex 2D and 3D structures, including curved nanostructures. In 2012, DNA origami was used to put together complex nanostructures such as gated transmembrane channels.9,10That same year, an approach for the design of complex 3D DNA structures was reported based on the assembly of short oligonucleotides (without a scaffolding strand) termed DNA bricks.11 The key to this approach lies in linking structural units (8-mer) to physical location, in effect creating an eight-nucleobase address for each position in a discrete 3D space. The use of unique addresses made it possible to assemble 3D structures based on predesigned sets of short DNA oligomers (“bricks”)—each DNA brick a 32-mer, designed as four contiguous eight-base domains targeting adjacent physical addresses, reminiscent of a 2 × 1 LEGO® brick.Although not all solutions have been released on the market, long-range DNA synthesis is clearly possible and improving in terms of price, speed, and quality. And there is an increasing number of solutions in all customer segments that seek to shape the future of biotechnology using long synthetic DNA.The development of DNA synthesis technologies may also be relevant in materials science and nanotechnology. Nucleic acids are a remarkably versatile source of novel materials and nanodevices. Design and evolution combined with the sub-angstrom resolution offered by nucleic acid scaffolds may enable not only the assembly of intricate structures but also confer them with properties such as tight ligand binding, catalytic activity, and different conformational states. Chemical modification of nucleic acids, and in particular wholesale replacement of the natural framework with synthetic genetic polymers (xeno nucleic acids [XNAs]) capable of evolution, has the potential to create novel structures and devices as well as greatly extend their functionalities—for instance, generating XNA devices that are stable in vivo and that can be used in diagnostic applications capitalizing on fluorescence or catalytic properties.Ongoing work to expand the chemistry that is compatible with enzymatic synthesis and evolution will add to the expanding palette of XNAs available for the construction of XNA nanostructures and devices enabling novel functions and improved performance in challenging environments and conditions.

Anzalone Prime: An Interview with Prime Editing Developer Andrew Anzalone

GEN BiotechnologyVol. 2, No. 2 Asked & AnsweredFree AccessAnzalone Prime: An Interview with Prime Editing Developer Andrew AnzaloneAndrew Anzalone, Alex Philippidis, and Kevin DaviesAndrew Anzalone*Address correspondence to: Andrew Anzalone, Prime Medicine, 21 Erie St, Cambridge, MA 02139, USA, E-mail Address: aanzalone@primemedicine.comPrime Medicine, Cambridge, Massachusetts, USA.Search for more papers by this author, Alex PhilippidisSenior Business Editor, GEN.Search for more papers by this author, and Kevin DaviesGEN Biotechnology, New Rochelle, New York, USA.Search for more papers by this authorPublished Online:18 Apr 2023https://doi.org/10.1089/genbio.2023.29091.aanAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Andrew Anzalone, Co-Founder and Head of Prime Editing Platform at Prime Medicine.Andrew Anzalone is the lead developer of prime editing (PE) and the scientific cofounder and head of the PE platform at Prime Medicine. Founded in 2019, Prime Medicine is one of several biotech companies developing new precision genome editing platforms capable of engineering a variety of base substitutions and other more flexible insertions and deletions.PE is an exciting technology for precision genome editing, because it can in principle engineer any potential nucleotide substitution by performing an ingenious RNA-based chemistry on the double helix itself (Fig. 1).FIG. 1. Programmable correction of genetic mutations with prime editing. In this example, a Prime Editor protein containing a Cas domain and a reverse transcriptase (RT) domain complexes with a prime editing guide RNA, or pegRNA (orange), that contains “search” and “replace” sequences. The Prime Editor complex is programmed to target a gene containing a mutated sequence (red) using the pegRNA's “search” sequence. After locating the mutated sequence, the Cas domain nicks one of the two DNA strands, liberating a 3’ DNA flap. The 3’ flap then pairs to a complementary portion of the pegRNA's “replace” sequence, allowing the RT domain to copy the programmed edit (blue) directly into the gene using the pegRNA's “replace” sequence as a template. Cellular DNA repair processes remove the mutated DNA sequence and incorporate the edited sequence into the complementary strand of DNA, resulting in correction of the mutation in both strands of DNA. (Courtesy: A. Anzalone/Prime Medicine.)In this interview, conducted by GEN Senior Business Editor Alex Philippidis and GEN Biotechnology Executive Editor Kevin Davies, Anzalone discusses the genesis of the PE technology and the clinical programs outlined by his colleagues at Prime Medicine.(This interview has been lightly edited for length and clarity).GEN Biotechnology: Andrew, it has only been about 5 years since your time at Columbia University, where you got your MD and PhD, and the inception of PE technology. How did it all begin?Anzalone: It all started back in my days as a trainee, where I was focused on chemistry and molecular engineering. I was also very interested in medicine and seeing how we can push science forward to help people with diseases. As I went through my training, I had such an enthusiasm for science, working in the laboratory, and this desire to develop therapies. As I moved toward the end of my training, I wanted to look for a research position that would allow me to really translate this science into something potentially meaningful.In the backdrop of all this, of course, was this CRISPR revolution that had started around 2012–2013, with some landmark publications from Jennifer Doudna, Emmanuelle Charpentier, Feng Zhang, George Church, the list goes on and on.As I looked for new opportunities in my research and new directions, I came across some really interesting work in this gene editing field using these CRISPR technologies. And David Liu (Broad Institute) quickly rose up on my list of people of interest. He had just developed a base editing system, which could precisely make single base-pair changes to DNA. Not only did he develop one base editor, but he had also just published a second base editor, about a year apart from each other.I looked at this, and I thought, “Wow! This is really exploding.” This is something that I could have never imagined would be possible 10 years ago, when I had started medical school, learning about all these different genetic diseases. So I saw that as an opportunity to build on what had been done prior. We have this incredible natural system, these CRISPR enzymes that can target, in a programmable way, a DNA sequence, to go and find it in this vast sea of otherwise similar looking DNA sequences.And we could start making more sophisticated machines that could edit the DNA in different ways. Of course, a big challenge was: we have this CRISPR system that can target and find sequences in the genome, but how to change them to exactly what you want was still a bit of a challenge. There was still something to be done to address the second half of what was required for gene editing. That is where the idea came from for how we might develop a new system, which eventually became PE.An earlier report said your idea came on an evening stroll around your New York neighborhood. It is not exactly Kary Mullis popping LSD on a midnight drive through Napa Valley en route to discovering polymerase chain reaction, is it?! Perhaps you can reveal how the real breakthrough came about?Yes, there was no LSD involved! But I do like to do my thinking on walks, when I can do some hard thinking. At some point, a notion occurred to me that these CRISPR systems are so elegant—they have these RNA molecules that are almost like instructions and give them an address for where to go in the genome, to bind to DNA, and then to change that DNA. What a nice opportunity for that programmability to now not just be used to say where to go, but also what to do.In fact, we have already got this RNA sequence that has information in it, bound at the target side in the genome where you want to make a change. Why not just add some instructions on what that change might be? We have got programmability for targeting. What about the programmability for editing? The system is already poised to do that. It already has the right molecules in place. So that was the conception. Obviously, that is just an idea that is not worth much on its own. It was not until I entered the Broad Institute that I really started to get into the details and think about how this might be possible.Did you have to twist David's arm a little bit?I think many people saw the promise in the idea, including David, but also many of the challenges that we would face. There were many questions that, in any one of them, if things did not go as planned, the system would not work so well. There was some optimism, [but] a healthy amount of skepticism as well. It is a bit of a funny story. I had discussed the idea on my postdoc interview with David and many members of the group, and it was quite a colorful discussion, but a really nice one. A few weeks after my interview, I followed up with David and he agreed to allow me to join the laboratory and pursue this project.Initially, it started as a project that only I worked on. I cannot emphasize enough how great an environment David had developed in his laboratory, in terms of learning all the techniques from people. These were all things I had not done before. I had never done genome editing in a human cell before getting to David's laboratory, I had never done high-throughput sequencing. I definitely credit all my laboratory mates there for showing me these things and helping me talk through ideas to make this work.As the project showed more promise, others started to join me on the project. Ultimately the article we published had 12 or so coauthors on it, all of whom made a very important contribution.1 I really do credit the environment that was cultivated there to a lot of the success, and especially the speed of success.Was it smooth sailing that first year or did you have to overcome stumbling blocks?There were at least four challenges that laid in front of us. David refers to it as “four little miracles” that needed to happen for PE to actually work. When I had these little stumbling blocks we tweaked some things, worked on some new designs, and got past each one of them, one at a time until ultimately getting into human cells and making it work. Base editing is excellent at making certain categories of substitution, but PE, in principle, could give you the full repertoire, exploiting all there is to offer from having a nucleic acid sequence template.RNA and DNA share pretty much the same letters. That information is easily exchanged in both directions. The directions that we needed to exchange were from RNA into DNA —that is a process performed by a category of enzymes—reverse transcriptases. They synthesize DNA from an RNA template.Because this template sequence could be changed very simply by changing one base here, another base there, deleting some bases, adding some bases—that template will dictate what sequence goes into the DNA. So, it is easy to swap out one for another and make any edit in principle that you really want in a pretty confined but substantial region of DNA.Your landmark article, published in Nature1 (Fig. 2), was in press when David presented the work for the first time, at the Cold Spring Harbor CRISPR conference in 2019. Do you recall what he said at the end of his talk in his acknowledgment slide?FIG. 2. Prime editing debuted in this 2019 Nature paper by Anzalone and colleagues from the Broad Institute.It was a very meaningful experience for me. David said something along the lines of he is “excited to see what I do in the second year of my postdoc!” It was partially set back by the pandemic, unfortunately, but I think we still got a bit more done in the PE space then.By the time the Nature article was published we are talking toward the close of 2019, were you and David already thinking that this technology would be commercially developed as an independent company?Absolutely. There was a lot of recognition early on, a few months before we published the Nature article, because we were trying all these different things and they were all working at once. We finally developed the system, getting it to the state where it was working in human cells. We could swap out the RNA with a new sequence and make an edit somewhere else in the genome, and we just saw the potential for that. It does not take a lot of effort to look through the types of mutations that cause genetic disease and just tally up the percentage of those we could potentially fix with PE.It is a pretty large number, somewhere close to 90% of the mutations in certain databases such as ClinVar. We immediately recognized the opportunity there and the potential. And David obviously has a lot of experience founding companies and was able to get that going pretty efficiently.What are some of the advantages of PE beyond only nicking the double helix? Another is the expanded range of edits it can give researchers?What would an ideal gene editing system look like? There are probably five factors that many of us have discussed as being really critical. First is programmability. Essentially, this means you can go one place in the genome and then the next day change that location and go somewhere else and do that with ease and quickly.Second would be specificity. You only want to go to the place you want to go, not other places—that has implications for things like off-target editing. The third one is versatility. When you get to that place in the genome, can you make all the different types of changes you might want to make? How broad can that genome editing system be in terms of making a point mutation, deletion, etc.? And then how precisely will it do so at that location? Are you making just that change, or are you making other changes as well? Finally, how efficient is that system? Ideally, every cell that gets treated with these editing systems would make an edit.So these are five different principles that you would look for in an ideal gene editing system. PE, and in many ways all CRISPR systems, is very programmable. It is very straightforward to give a new set of instructions to these CRISPR guide RNAs, to go to a new place in the genome.Say one day, I want to fix this mutation in CFTR that causes cystic fibrosis. On another day, I want to fix this mutation in sickle cell disease. That is a real powerful principle of the CRISPR technology. Now, once you get there, you want to minimize the other things you do besides making the precise edit that you are intending to make. In a PE system, we are using this RNA template to make a change in principle. The fidelity of copying that sequence is very high. So you are very likely to only incorporate that one change and you only make a single break in the DNA.That is really important. When a cell has a double-strand break (this is what the more traditional CRISPR nucleases do), it sets off a lot of alarm bells. It quickly identifies that broken DNA end and stitches it together as fast as possible, without a lot of care for what happens to the sequence when it does that. So you can imagine these two pieces coming together. But little errors happening in that process lead to these small changes, insertions, or deletions of a few bases that often will disrupt a gene.That is just an intrinsic thing the cells will do. Now, if you make only one break in the double helix, you avoid many of the responses in the cell, and that is exactly what PE does. It makes just a single nick to copy DNA sequence onto that one strand of DNA and then other DNA repair mechanisms can incorporate that edit into both strands eventually. But you avoid alternative DNA repair pathways that tend to lead to undesirable outcomes.Another advantage is that at any potential off-target sites in the genome (places where you did not intend that prime editor system or CRISPR system to go to), you are not going to be making double-strand breaks, so it is much less likely to cause damage at those other places in the genome.When Prime Medicine launched in 2019, did you have any doubts about being an active part of building the company? Presumably you could have had your pick of any faculty position or maybe return to medicine?I thought hard about that but actually not that long. I had always been motivated from early days in research. Actually, my first research experiences were in medicinal chemistry at a small pharmaceutical company, so I was always interested in developing therapeutics.Obviously, the Liu laboratory had a lot of success doing science that had the ability to translate into therapeutics in the past, and that was one of the reasons I was very attracted to going to this group. When the opportunity (to join Prime Medicine) arose, I did not think too long. This would be a great opportunity for me. I love the science. I was there when it started. I cannot think of anything better than the possibility of being able to carry that forward to the point of using it for human therapeutics. That is really what drove me to join the company.In the first 6 months, the company's growth was a little slow—the early days of the pandemic. Those presented some challenges, but in the next 1–2 years, we have had an amazing growth period. We have hired up to almost 200 people now and we have been able to tap into the excitement that the community feels about gene editing and about PE in particular. Many staff members who have joined Prime Medicine have worked on gene editing in the past and have experience using PE. They have seen the potential and want to contribute to making it into medicines.Despite the obstacles, the pandemic, and competition and other things, we still had a pretty rapid growth trajectory, and brought in the people to get the job done. I am really proud about the team we have built.Who are some of the key members of your executive team, and is David Liu still actively involved?David is involved. He touches base with us on a weekly basis or so and gives us a lot of feedback on what we are doing. Also, his laboratory is actively working on PE technology development, so things that his group develops and the improvements to the technology often come over to us, and we have the ability to use those and capitalize on those developments.We have an excellent leadership team. CEO Keith Gottesdiener has been in biotech and pharma for several decades. Jeremy Duffield, our chief scientific officer, is also very experienced and an expert in the disease biology. We have put together a great group and have a lot of internal expertise.There are now six other people from David's laboratory at Prime Medicine who have been there from the beginning. All are authors on a PE publication, so this expertise has really helped us accelerate.In 2022, you filed a successful initial public offering (IPO) and released details of the pipeline. Why such an early IPO given how challenging the financial climate was?I think our ability to do the IPO probably signifies one thing—the excitement for the technology and its potential. It was an amazing experience to be part of that process to go public. I had to pinch myself a few times in Times Square in New York City. I have walked through there so many times in my life. I never thought I would be in the Nasdaq building, so it was quite the experience. I am really proud of the team for getting us there!We released our pipeline, and we have many areas that we are looking to work in. We were not sure when we started where PE was going to work or where it would work the best. Maybe there would be some places where it just would not work, and we would not be able to do anything in that area. But the more things we tried, we found things that work through the optimizations and enhancements to the technology that have been developed. Most of the areas where we are trying to do something, we have had a lot of success.Prime Medicine's pipeline is ambitious, featuring a dozen targets including well-known Mendelian diseases, including Duchenne muscular dystrophy and fragile X. What are some of the initial clinical priorities?That is a very nuanced question in the sense that we have the potential to address some of these really well-known diseases that have affected so many people. For many of them, there has not been a great therapeutic option available, so PE offers a really exciting opportunity. But in some cases, there are also challenges associated with delivery of the gene editing systems to the right cells in the body. So we have looked at the landscape and said, “there are certain places where we think we could be really successful early on, because some of these challenges have been addressed, such as delivery, and other places where we think the technology does something really special, but we probably need some other areas to catch up before they can become a possibility.”We have tried to develop a pipeline that has some examples from each of these categories. There shall be places where we can move a lot quicker. For example, ex vivo programs are a lot easier to do in the sense that the delivery challenges are not as prominent, and then certain in vivo programs where delivery has also been established previously.Are you trying to pursue all these programs yourselves or will you be seeking partners to develop some of them?It would be a little unrealistic to say that we are going to take 18 programs forward in the next few years and be able to develop them into medicines for people! I assume there are many solutions to those challenges, whether that be through partnering with other companies, or by prioritization, internally. That is something we will be deciding in the next couple of years.Which of these programs are furthest advanced and what is going to determine which targets Prime Medicine prioritizes?Those questions are still being worked through. The targets where we have had a lot of ability to deliver the gene editing systems to the cells of interest. Again, the ex vivo programs have the ability to get past that a little bit more quickly. Those we can make pretty rapid progress in, and likewise delivery by nanoparticles to the liver. These are also areas where we can move a little faster. That said, we are still paying a lot of attention to those other programs where there is still so much potential, they just may require other delivery solutions.The development of CRISPR in 2012–2013 led to a protracted patent dispute. Is there a possibility something similar could happen in the world of PE?I am certainly not a patent attorney! I will say that I believe Prime Medicine has a very strong position in PE. We filed patents at the Broad Institute very early on that cover the PE system. We have been awarded some of those patents, and we continute to file more patents on both our technology and our therapeutic applications. Additionally, we maintain a strong relationship with David Liu's group and have in-licensed their PE developments as well. So I feel very good about our intellectual property position and the work that we did to establish that position.There is increasing competition in the development of a new generation or new wave of CRISPR-based gene editing tools—PASTE, twin PE and transposases have had a lot of attention. Do you anticipate increased competition in developing these precision editing tools as we go forward?The CRISPR space has always been competitive and genome editing in general has been a very competitive space. Even from the beginning, when the first CRISPR systems were described, there were races to find new effector proteins, new CRISPR systems that could be translated into therapeutics, whether those be smaller Cas systems or others that had other beneficial properties. Similarly, there has been competition to develop the next generation approaches to do genome editing, which has been very healthy, making this technology more effective and ultimately more useful for the development of therapeutics. I think that is a really exciting thing to witness as a scientist and as a member of the community.From the standpoint of Prime Medicine, we are also really interested in continuing to do that development and contributing to that innovation and moving things forward.You are attacking a wide array of genetic disorders including some that are not simple base substitutions. What is it about the platform that allows you to potentially tackle some of these more complicated genetic mechanisms?One of the developments to the PE technology that I have not discussed yet is “dual flap prime editing” or twin-PE. Essentially this is an approach to PE that allows you to replace or delete much larger pieces of DNA. One of the exciting applications for that, in my opinion, is the ability to delete expanded repeats in DNA that are known to cause some of these diseases—Huntington's disease, Fragile X, and Friedreich's ataxia. There is a long list of these (repeat expansion disorders), they are devastating and very challenging to treat. So PE has this second-generation dual-flap version that can potentially be harnessed to address some of these.Beyond that, we are very interested in other larger changes that you could make to a genome, including the insertion of large DNA sequences. With the twin-PE approach, we showed when I was still in David's laboratory that you could use it to variously and efficiently insert short DNA sequences of 30–50 bases that are called recombinase attachment sites. These can be paired up with a class of enzymes called integrases or recombinases. Many scientists have used versions of these to create companies.Once that specific attachment site has been prime edited into a specific location in the genome, these recombinases can take new DNA pieces and insert them there. We have developed the mechanism for targeted integration of large pieces of DNA. We did the proof of concept in the Liu laboratory and we have been able to develop it at Prime Medicine. At the JP Morgan conference, we showed an example of this in T cells, where we could do targeted integration of 3.5 kb DNA at 60% efficiency.All these additional developments on that PE platform are enabling so many new things, and allow us to address new exciting areas that the initial PE system maybe would not have been able to do.There are many other factors in successfully developing a gene editing technology besides optimizing the chemistry, including delivery. To what extent is Prime Medicine exploring other important aspects of building a successful clinical platform?We are very invested in that. We are here to develop medicines, not to do science projects that can only be demonstrated in a tissue culture experiment. Of course, that is the beginning, but it is not the endpoint. As you have mentioned, delivery approaches are required to deliver these therapeutics, as is (bone marrow) conditioning and other associated processes. This is all part of making the medicine, so we are very much thinking about that on the delivery front.We have been thinking a lot about how do we get our gene editing system into cells in vivo, using transient delivery systems that are based on RNA. These are lipid nanoparticle formulations, the same way that many of the COVID-19 vaccines have been delivered.We are thinking about ways that we can get these into other cell types beyond the liver, which is where primarily they have been effective, and then also thinking of viral delivery solutions that can get our gene editing components into even broader sets of cell types, such as the central nervous system. We are attempting to develop a full medicine, not just the gene editing system.Some genetic therapies that have been recently approved have price tags of $2–3 million or more. You went into training to be a doctor. How do you balance the need for these biotech companies to get a return on their huge investments with delivering an affordable therapy for millions of patients around the world?It is an important question. There are these therapies that could be developed for the Western world, and genetic diseases affect everybody and not in the same way. Obviously, these are inherited from family members and (affect) a large percentage of patients. Certain regions of the world have their own sets of genetic mutations that you might have to fix, so developing treatments for everybody—we are going to figure out ways to pay forward and make it accessible to patients.In the near term, my personal view, which is not the most sophisticated, is that that is going to require—just based on the amount of investment and time that it takes to make one of these the first time—that the cost be proportional, so that we can support the growth of this new therapeutic area. My hope would be that over time, we learn how to do this with more ease, with less upfront investment, with more of an ability to swap out a guide RNA, for example, and not have to do an entirely new clinical trial and be able to move from one patient and their mutation to another patient and their mutation with very minimal extra cost. I think, as this world comes into reality [we will see], the possibility of making these genetic systems for more patients at a very affordable price would make sense.I cannot tell you how many times I saw patients come into the hospital for the same problem over and over again, because of their genetic disease. And from a cost standpoint, it might even make sense to treat that patient, and prevent all those recurrent costs, not to mention the quality of their life that would be affected, and most importantly how much of a benefit that would have to them. I think there is a good reason to invest here, and from my personal perspective, I hope that costs can be minimized in the future so that this is not a major restriction.Reference1. Anzalone AV, Randolph PB, Davis JR, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 2019;576:149–157; doi: 10.1038/s41586-019-1711-4 Crossref, Medline, Google ScholarFiguresReferencesRelatedDetails Volume 2Issue 2Apr 2023 InformationCopyright 2023, Mary Ann Liebert, Inc., publishersTo cite this article:Andrew Anzalone, Alex Philippidis, and Kevin Davies.Anzalone Prime: An Interview with Prime Editing Developer Andrew Anzalone.GEN Biotechnology.Apr 2023.81-86.http://doi.org/10.1089/genbio.2023.29091.aanPublished in Volume: 2 Issue 2: April 18, 2023PDF download

PASTE, Don't Cut: Genome Editing Tool Looks Beyond CRISPR and Prime

GEN BiotechnologyVol. 2, No. 2 News FeatureFree AccessPASTE, Don't Cut: Genome Editing Tool Looks Beyond CRISPR and PrimeAlex PhilippidisAlex PhilippidisE-mail Address: aphilippidis@genengnews.comSenior Business Editor, GEN.Contributing Editor, GEN Biotechnology.Search for more papers by this authorPublished Online:18 Apr 2023https://doi.org/10.1089/genbio.2023.29088.aphAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail MIT McGovern Institute researchers Omar Abudayyeh and Jonathan Gootenberg look to expand the universe of diseases treatable via genome editing. Will their patent touch off another legal wrangle for the field?A recently patented genome editing tool called programmable addition via site-specific targeting elements (PASTE) holds genuine promise for expanding the universe of treatable genetic diseases. The approach combines elements of CRISPR and prime editing with a pair of enzymes designed to enable the integration of large segments of DNA without incurring double-stranded DNA breaks.US Patent No. 11,572,556, assigned to MIT, covers systems, methods, and compositions for PASTE. The patent describes site-specific integration of a nucleic acid into a genome, using a CRISPR–Cas9 nickase fused to a reverse transcriptase (RT) and a serine integrase. These enzymes target specific genome sequences known as attachment sites, binding to them before integrating their DNA payload (Fig. 1).FIG. 1. PASTE editing uses a recombinase-prime editor and a pegRNA encoding the attachment site (termed atgRNA) to enable a two-step editing procedure in which attB is inserted first followed by donor DNA integration. (Adapted from Mahmood and Mansoor8). pegRNA, prime editing guide RNA.Invented by Omar Abudayyeh and Jonathan Gootenberg, a pair of McGovern fellows at MIT's McGovern Institute for Brain Research, PASTE can insert DNA fragments as large as 50 kilobases (kb). This places PASTE on a different plane compared with other genome editing tools such as prime editing.Abudayyeh and Gootenberg were both graduate students with CRISPR pioneer Feng Zhang, at the Broad Institute before moving across the road to the McGovern Institute in Kendall Square, Cambridge, MA, to set up their own joint laboratory in 2019. They first detailed PASTE in a preprint posted on bioRxiv in 2021. The peer-reviewed article was published in November 2022 in Nature Biotechnology.1Omar Abudayyeh (top) and Jonathan Gootenberg (bottom) are McGovern fellows at MIT's McGovern Institute for Brain Research.PASTE entails the engineering of Cas9, RT, and integrase linkers to create a fusion protein capable of efficient integration (5–50%) of diverse cargos at precisely defined target locations within the human genome with small stereotyped scars that can serve as protein linkers. The serine integrases used in PASTE can insert large DNA sequences by targeting specific “attachment” sites within the genome.“It has been very difficult for the [CRISPR] field to really put large edits in the genome without relying on things like homology-directed repair,” Gootenberg told me. “The concept of PASTE is actually quite simple: Instead of being able to try to do everything all at once in one conceptual aspect—and there are some technologies that approach that—we thought it would be easier to take something that can insert very efficiently into a constant sequence like an integrase and then put the constant sequence into the genome.”“It's a two-step process, where we first insert the constant sequence, which is a small insert—the site is between 38–46 nucleotides long—and it's easier to do. Then we use that constant sequence to put in a larger sequence. That's the concept in a nutshell,” Gootenberg added.The $64-Million QuestionJacob S. Sherkow, a professor at the University of Illinois College of Law and the Carle Illinois College of Medicine, and an authority on the long-standing CRISPR–Cas9 patent standoff,2 says that PASTE appears to address a key challenge in genome editing: How to achieve high fidelity site-specific insertion of an exogenous target sequence?Citing the three-prong standard for patentability—novelty, utility, and nonobviousness—Sherkow said MIT and the PASTE inventors can likely show their technology is novel and useful. But it is less clear if they can show nonobviousness should the PASTE patent be challenged someday. “That is the $64-million (sic) question,” said Sherkow. “To the extent that there's patent litigation, I assure you that on the PASTE patent, at least some of those arguments are going to turn on non-obviousness.”David Liu, The Broad Institute and Harvard University; Co-Founder of Prime MedicineJacob Sherkow, University of Illinois“You've got the older [David] Liu prime editing patent that claims prime editing. Then you've got [the] PASTE patent filed later, which claims 80% of what the prime editing patent claims, plus the addition of using an interface and an RT, to insert a large piece of DNA in a site-specific site.”“That is not an overlap issue at all, such that the patent office is going to be asked to, or have the authority to, cancel one patent in favor of the other,” Sherkow said. “Where the conflict is going to arise is not on the validity of these two patents. It's going to be on who is going to pay for them and who is going to use which technology over another technology.”Precision genome editing without the need for double-stranded breaks in DNA was first laid out by David Liu, and colleagues at the Broad Institute. In a pair of articles in Nature published between 2015 and 2016, Liu's laboratory developed base editing, a technique that can make certain classes of single-base substitutions without cleaving the double helix. Two former postdocs in Liu's laboratory, Alexis Komor (now on the faculty of University of California, San Diego) and Nicole Gaudelli, (Beam Therapeutics), led the development of base editing, which is being commercially developed by Beam Therapeutics.3Inspired by Liu's work on base editing, Andrew Anzalone conceived and led the development of an RNA-based “search-and-replace” genome editing technology dubbed prime editing, reported in a landmark 2019 article published in Nature.4 Prime editing can introduce targeted insertions, deletions, and all 12 possible nucleotide substitutions. Among roughly 75,000 catalogued pathogenic mutations in human genetic diseases, prime editing has the potential to correct up to 89% of them.Liu cofounded Prime Medicine (see Box 1) to commercialize prime editing based on Anzalone's groundbreaking work. (Anzalone is Prime Medicine's scientific cofounder and head of its prime editing platform; see the interview with Anzalone5 in this issue.)Box 1. Search and ReplaceLast year, Prime Medicine completed an initial public offering that raised ∼$180 million despite the bear market for newly public biotechs. In its prospectus, Prime Medicine revealed an 18-program pipeline and the in-licensing of U.S. Patent No. 11,447,770, which covers methods of using prime editors, was granted on September 20, 2022, and expires in 2040.“We are confident in the strength of our patent portfolio,” Prime Medicine said in a statement. “Broad Institute's in-licensed IP includes an issued U.S. patent broadly covering [prime editing] methods and an allowed U.S. application, which is expected to issue shortly, covering pegRNAs.” Prime Medicine says its patent portfolio includes “numerous in-licensed and Prime Medicine-owned patent applications in the U.S. and worldwide.” Both the Broad Institute and Prime Medicine have filed for patent protection covering technological advancements that will “greatly” expand the scope of prime editing.In addition to offering the full slate of nucleotide substitutions, prime editing can in principle engineer insertions and deletions as well as alter the regulatory regions of genes, insert or create premature stop codons, and modify splicing sequences.Prime Medicine has and will likely continue to in-license prime editing improvements from Liu's laboratory. “We believe these investments, along with our continuing relationship with Dr. Liu, establish Prime Medicine as a clear leader in prime editing,” the company added. “We plan to continue… reinforcing our leadership position and making fundamental progress towards better therapies for patients.”Prime Medicine recently announced the selection of its first development candidate—PM359, a treatment for chronic granulomatous disease, after observing long-term in vivo engraftment of prime-edited hematopoietic stem cells in a mouse model of the disease.“We look forward to advancing PM359 into investigational new drug (IND)-enabling studies later this year, while continuing to advance our broader portfolio toward additional preclinical proof-of-concept readouts,” Keith Gottesdiener, Prime Medicine's President and CEO, said in a statement.In January, Prime Medicine announced positive preclinical data for three of its development programs—candidates to treat Friedrich's ataxia, cystic fibrosis, and Wilson's disease. In addition, Prime Medicine said its PASSIGE platform achieved an ∼60% precise insertion of a 3.5-kb transgene at a single target site in primary human T cells, resulting in positive expression of the gene product.The 2019 article showed prime editing being used to install an integrase/recombinase landing site into a target DNA site. Earlier, in March 2019, Liu and colleagues filed for a U.S. patent that was granted in September 2022 (No. 11,447,770), which described several examples using prime editing to install integrase/recombinase landing sites, followed by targeted gene integration using an integrase/recombinase enzyme. In 2021, Liu's team published the use of “twin” prime editing to install an integrase/recombinase landing site into a target DNA site, followed by the catalytic integration of cargo DNA into that landing site.6 Prime Medicine is pursuing this technology, which it calls PASSIGE™ (prime-assisted site-specific integrase gene editing).PASTE Versus PASSIGELiu explains that one distinction between PASTE and PASSIGE is that PASTE fuses the prime editor and the integrase enzyme into a single molecule, whereas PASSIGE typically uses two separate proteins. Liu's verdict is that “the two proteins must act separately.”“We have compared, side-by-side, fused and unfused prime editors plus integrases at several different target sites in human cells, and we have never observed any benefit from fusing the prime editor and integrase,” says Liu, who is also the director of the Merkin Institute of Transformative Technologies in Healthcare and a Howard Hughes Medical Institute investigator.“Indeed, for most of the target sites and integrase enzyme combinations we've tested in human cells, we observed that the fused prime editor–integrase proteins as reported in the PASTE paper substantially underperform the separated prime editor and integrase proteins as used in PASSIGE,” Liu said. The improved performance of separate prime editor and integrase proteins, rather than fusing the two proteins together, makes sense scientifically, Liu says, “because the prime editor must vacate the target landing site before the integrase enzyme can perform cargo DNA integration.”According to Liu, fusing the two proteins increases the chance that the target landing site is blocked by the prime editing protein and/or associated prime editing guide RNA (pegRNA), “because the integrase must compete with an always-nearby prime editor to access the target landing site when the two proteins are tethered.”In contrast, Liu says, fusing the DNA-nicking Cas protein and the engineered RT together into a prime editor makes sense “because the Cas protein must hold open—or melt—the two DNA strands in order for the nicked target DNA strand to prime reverse transcription, initiating the prime editing process,” Liu added. Fusing the Cas nickase to the engineered RT keeps the engineered RT nearby, “poised to act on the opened and nicked target DNA site.”Light Bulb MomentAsked about the potential for a challenge to the PASTE patent, Gootenberg acknowledged: “It's obviously going to be a complex situation with intersecting IP, as has been with Cas9 and other nucleases from the start. But that said, we're really excited about how we can develop these technologies and move these technologies forward to develop actual cures for patients.”Abudayyeh and Gootenberg are veterans of genome editing, having previously discovered Cas13 in the laboratory of Zhang, and subsequently in their own laboratory, the CRISPR effector protein Cas7–11. They shared a long-standing interest in targeted gene integration. “When we saw the [Anzalone] paper, a light bulb went off in our heads!” Abuddayeh recalled. “We saw these integrases that we were familiar with already. If we could somehow combine these systems together, [we could] basically use them to lay (at) the target site, too.”“When we first started down this path, Cas9 was the only thing people were focusing on for programmable double-stranded cleavage and editing. We asked the question of whether we can go beyond Cas9,” Abudayyeh added.Gootenberg and Abudayyeh have licensed the technology behind PASTE editing to a startup they cofounded, Tome Biosciences, which has reportedly raised more than $95 million from several big-name investors, including a16z, ARCH Venture Partners, Polaris Partners, and Google's GV. (The company is still in stealth mode.)“Were it not for the weak market, I suspect (the PASTE patent) would have propelled CRISPR-related stocks higher. But this also demonstrates that—while markets are weak—innovation in the space is still going strong,” Jeff Brown, founder and chief investment analyst for Brownstone Research wrote last September.Prime editing and PASTE are among numerous technologies that have developed in the rapidly evolving genome editing field as researchers and startups pursue curative therapies that engineer changes to the genome without introducing double-stranded DNA breaks. Last year, Intellia Therapeutics shelled out up to $200 million to buy Rewrite Therapeutics (a University of California, Berkeley startup) and its DNA writing technology.Patentability is among the key challenges for genome editing. For example, Tessera Therapeutics is pursuing a patent for its RNA gene writing technology, which uses a mechanism called target primed reverse transcription to write genes into the genome.7 The method involves four steps—binding RNA, binding DNA, nicking DNA, and priming reverse transcription. The company is among several whose intellectual property has been challenged, according to a recent article in STAT, as “bear[ing] strong similarity to prime editing—even if they use different terminology.”Blanket TherapyPASTE editing could potentially treat diseases caused by genes harboring a large number of mutations, such as Leber's congenital amaurosis or cystic fibrosis, where gene editing systems would have to be tailored for specific mutations and each subset of a disease population. In those diseases, programmable insertion of a wild-type gene could address most potential mutations and serve as a blanket therapy.“Complete replacement of genes at their natural sites can be contemplated instead of creating variant-specific treatments,” Muhammad Arslan Mahmood and Shahid Mansoor, both of Pakistan's National Institute for Biotechnology and Genetic Engineering, observed in a recent commentary published in The CRISPR Journal.8“These exciting results enhance the versatility of the CRISPR-based gene editing along with the LSRs [large serine recombinases] that provide the opportunity for genome engineering, combinatory screening of bulky DNA libraries aiding to spread these applications for treating the diseases in both animals and plants, which are caused by defective genes,” the authors concluded.The Cystic Fibrosis Foundation is among the entities that have funded PASTE editing research, through a pioneer award designed to fund “ambitious basic research projects aiming to utilize cutting-edge techniques and strategies that have the potential to discover new genetic-based therapies for cystic fibrosis.”“We've been working on actually being able to insert the genes at the locus, and things are looking promising… That's one of the main funded applications we're looking at right now,” Gootenberg said.On a RollFeng Zhang's entrepreneurial spirit appears to have rubbed off on his two proteges. In addition to Tome, Gootenberg and Abudayyeh have also cofounded Sherlock Biosciences, which recently gained rights to a U.S. patent for diagnostic use of a CRISPR system based on Cas12; Proof Diagnostics, a CRISPR-based COVID-19 molecular test developer; and Moment Biosciences, a precision microbiome therapy developer.In the 2022 PASTE study, Abudayyeh, Gootenberg, and colleagues described delivery of genes ranging from 779 to 36,000 base pairs—a range that would enable insertion of >99.7% of human cDNAs as transgenes—to a variety of human cell types. Tests with more than a dozen payload genes revealed insertion success rates ranging from 5% to 60%, with “minimal” formation of indels at nine different integration sites.The researchers also reported inserting genes in “humanized” livers in mice, consisting of ∼70% human hepatocytes, with PASTE showing a much lower percentage (as high as 2.5%) of successfully integrating new genes—with average insertion ranging from 0.3% to 1.4%.Can PASTE transport genes larger than 36 kb? “We've not done larger, but there's no reason I think why you couldn't do larger,” Abudayyeh said. “It becomes a delivery problem: How do we get [50-kb] templates into a human cell? The reason we're able to max out at approximately 36 kb is because we're using an adenoviral construct. Adenoviruses are approximately a 36-kb genome, so it's easy to get that into the cell.”Abudayyeh said they have been approached by researchers interested in testing inserts of 50–100 kb. “We just haven't gone down that path,” he said. On its minimalist website, however, Tome Biosciences suggests it can handle DNA segments of that size range: “Using CRISPR, our technologies allow us to insert any genetic sequence of any size at any location into any genome.”Beyond Patent ValidityAlthough users of prime editing do not likely need a license covering PASTE, the converse might not be true. Sherkow states: “By contrast, if you are using the PASTE technology, there is, I think, an outstanding question as to whether you will also need to obtain a prime patent license.”How companies answer that question may well depend on how they plan to use both PASTE and prime editing technology. “This is more just, frankly, garden-variety technology, improvement licensing stuff that happens in a variety of other fields,” Sherkow said. “It is not this cataclysmic dispute with respect to who got their first and validity, the way that we had in the CRISPR 1.0 context.”Sherkow has written extensively on the twists and turns in the long-running CRISPR–Cas9 patent saga. In that case, he says, “in some conceptual sense either the [University of California] patents are valid and the Broad Institute's are not, or vice versa. That's just not necessarily true here between Harvard with prime editing and MIT with PASTE,” Sherkow said. “One could be valid, one could be not. One could be infringed, one could be not.”“It's going to come down to whether a company that wants to develop a product in this area is going to be counseled to take a license to one or the other, or both. I think that's going to be a function of what they think the best fit for their technology is,” Sherkow added.As a result, he believes researchers will be inclined to use prime editing or PASTE—and thus pursue licenses for one or the other—based on the diseases they are working to treat. “Unlike the CRISPR 1.0 situation, where who are we going to take a license to turns on, who do we think is going to win the patent fight, taking a license for prime [editing] or PASTE turns on what is our actual technology, which to be blunt is how we want the licensing system to work,” Sherkow said.“We don't want the licensing system to work where people are placing horse bets on winners,” says Sherkow, “while others are just losing their shirts.”A version of this story was originally published online by GEN.