Genome Engineering Approaches Research Articles

Making gene editing accessible in resource limited environments: recommendations to guide a first-time user

The designer nuclease, CRISPR-Cas9 system has advanced the field of genome engineering owing to its programmability and ease of use. The application of these molecular scissors for genome engineering earned the developing researchers the Nobel prize in Chemistry in the year 2020. At present, the potential of this technology to improve global challenges continues to grow exponentially. CRISPR-Cas9 shows promise in the recent advances made in the Global North such as the FDA-approved gene therapy for the treatment of sickle cell anaemia and β-thalassemia and the gene editing of porcine kidney for xenotransplantation into humans affected by end-stage kidney failure. Limited resources, low government investment with an allocation of 1% of gross domestic production to research and development including a shortage of skilled professionals and lack of knowledge may preclude the use of this revolutionary technology in the Global South where the countries involved have reduced science and technology budgets. Focusing on the practical application of genome engineering, successful genetic manipulation is not easily accomplishable and is influenced by the chromatin landscape of the target locus, guide RNA selection, the experimental design including the profiling of the gene edited cells, which impacts the overall outcome achieved. Our assessment primarily delves into economical approaches of performing efficient genome engineering to support the first-time user restricted by limited resources with the aim of democratizing the use of the technology across low- and middle-income countries. Here we provide a comprehensive overview on existing experimental techniques, the significance for target locus analysis and current pitfalls such as the underrepresentation of global genetic diversity. Several perspectives of genome engineering approaches are outlined, which can be adopted in a resource limited setting to enable a higher success rate of genome editing-based innovations in low- and middle-income countries.

Profiling Cas9- and Cas12a-induced mutagenesis in Arabidopsis thaliana.

With the advancement of CRISPR technologies, a comprehensive understanding of repair mechanisms following double-strand break (DSB) formation is important for improving the precision and efficiency of genetic modifications. In plant genetics, two Cas nucleases are widely used, i.e. Cas9 and Cas12a, which differ with respect to PAM sequence composition, position of the DSB relative to the PAM, and DSB-end configuration (blunt vs. staggered). The latter difference has led to speculations about different options for repair and recombination. Here, we provide detailed repair profiles for LbCas12a in Arabidopsis thaliana, using identical experimental settings previously reported for Cas9-induced DSBs, thus allowing for a quantitative comparison of both nucleases. For both enzymes, non-homologous end-joining (NHEJ) produces 70% of mutations, whereas polymerase theta-mediated end-joining (TMEJ) generates 30%, indicating that DSB-end configuration does not dictate repair pathway choice. Relevant for genome engineering approaches aimed at integrating exogenous DNA, we found that Cas12a similarly stimulates the integration of T-DNA molecules as does Cas9. Long-read sequencing of both Cas9 and Cas12a repair outcomes further revealed a previously underappreciated degree of DNA loss upon TMEJ. The most notable disparity between Cas9 and Cas12a repair profiles is caused by how NHEJ acts on DSB ends with short overhangs: non-symmetric Cas9 cleavage produce 1 bp insertions, which we here show to depend on polymerase Lambda, whereas staggered Cas12a DSBs are not subjected to fill-in synthesis. We conclude that Cas9 and Cas12a are equally effective for genome engineering purposes, offering flexibility in nuclease choice based on the availability of compatible PAM sequences.

Abstract NG03: Functional and computational approaches to uncover selection advantages of cancer aneuploidy

Abstract Aneuploidy, including the gain or loss of whole chromosomes or chromosome arms, is a near-universal feature of cancer. We previously applied methods that define chromosome arm aneuploidy to over 10,000 tumors in the Cancer Genome Atlas (TCGA). Cancer subtypes are often characterized by tumor specific patterns of chromosome arm copy number alterations and breakpoints; for example, squamous cell carcinomas (SCCs) from different tissues of origin are characterized by chromosome 3p (chr3p) loss and chromosome 3q (chr3q) gain. From the TCGA aneuploidy data, we developed an algorithm called BISCUT to distinguish peak regions of aneuploidy breakpoints on each chromosome arm. BISCUT identified loci affected by broad copy number alterations that provide fitness advantages or disadvantages both within individual cancer types and across cancers. Our analyses are consistent with selection being the primary driver of aneuploidy events in cancer. We next wanted to validate some BISCUT peaks without a known driver, to identify potential gene deletions that are beneficial in cancer cells. We focused on chromosome 8p (chr8p), as this arm is frequently deleted across cancer types, but no strong tumor suppressors have been identified. Recent advances in genome engineering allow generation of large chromosomal alterations and validation of findings from patient genomic data. For this study, we used our CRISPR-Cas9 arm-deletion system to delete chr8p in human immortalized epithelial cells. Cells with chr8p deletion showed lower amounts of cell death in culture. Knockdown of WRN, one of the two genes in the smallest chr8p BISCUT peak, was sufficient to reproduce this phenotype, suggesting that WRN haploinsufficiency may be beneficial to tumor development. As aneuploidy occurs in tissue-specific patterns, we also wanted to explore the role of chr3 arm aneuploidies in squamous cancers. We again used the CRISPR-Cas9 system to delete one copy of chr3p in a human immortalized lung epithelial cell line similar to the putative cell-of-origin in lung SCC. Consistent with patient data, expression of chr3p genes was decreased upon deletion, as well as increased expression of interferon response genes. Cells with chr3p deletion initially proliferated more slowly than their siblings. Interestingly, after several passages in culture, this proliferation defect was rescued in chr3p deleted cells. Genome sequencing and karyotype analyses suggested that this was partially the result of chr3 duplication, with cells transitioning to a state of chr3q gain. Culturing these cells as organoids also demonstrated that chr3p deletion and chr3q gain can affect basal cell differentiation, consistent with their high frequency in squamous cancers. Our genome engineering approach to model chromosome arm aneuploidies provides a robust model to validate drivers from aneuploidy events, which will be critical to address the gap in our understanding of aneuploidy in cancer. In addition, our methods have identified consequences of individual aneuploidy events, which will lead to new precision oncology targets for patients. Citation Format: Alison M. Taylor. Functional and computational approaches to uncover selection advantages of cancer aneuploidy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr NG03.

HIV-1 Proviral Genome Engineering with CRISPR-Cas9 for Mechanistic Studies.

HIV-1 latency remains a barrier to a functional cure because of the ability of virtually silent yet inducible proviruses within reservoir cells to transcriptionally reactivate upon cell stimulation. HIV-1 reactivation occurs through the sequential action of host transcription factors (TFs) during the "host phase" and the viral TF Tat during the "viral phase", which together facilitate the positive feedback loop required for exponential transcription, replication, and pathogenesis. The sequential action of these TFs poses a challenge to precisely delineate the contributions of the host and viral phases of the transcriptional program to guide future mechanistic and therapeutic studies. To address this limitation, we devised a genome engineering approach to mutate tat and create a genetically matched pair of Jurkat T cell clones harboring HIV-1 at the same integration site with and without Tat expression. By comparing the transcriptional profile of both clones, the transition point between the host and viral phases was defined, providing a system that enables the temporal mechanistic interrogation of HIV-1 transcription prior to and after Tat synthesis. Importantly, this CRISPR method is broadly applicable to knockout individual viral proteins or genomic regulatory elements to delineate their contributions to various aspects of the viral life cycle and ultimately may facilitate therapeutic approaches in our race towards achieving a functional cure.

Engineering approaches for RNA-based and cell-based osteoarthritis therapies.

Osteoarthritis (OA) is a chronic, debilitating disease that substantially impairs the quality of life of affected individuals. The underlying mechanisms of OA are diverse and arebecoming increasingly understood at the systemic, tissue, cellular and gene levels. However, the pharmacological therapies available remain limited, owing to drug delivery barriers, and consist mainlyof broadly immunosuppressive regimens, such as corticosteroids, that provide onlyshort-term palliative benefits and do not alter disease progression. Engineered RNA-based and cell-based therapies developed with synthetic chemistry and biology tools provide promise for future OA treatments with durable, efficacious mechanisms of action that can specifically target the underlying drivers of pathology. This Review highlights emerging classes of RNA-based technologies that hold potential for OA therapies, including smallinterfering RNA for gene silencing, microRNA and anti-microRNA for multi-gene regulation, mRNA for gene supplementation, and RNA-guided gene-editing platforms such as CRISPR-Cas9. Various cell-engineering strategies are also examined that potentiate disease-dependent, spatiotemporally regulated production of therapeutic molecules, and a conceptual framework is presented for their application as OA treatments. In summary, this Review highlights modern genetic medicines that have been clinically approved for other diseases, in addition to emerging genome and cellular engineering approaches, with the goal of emphasizing their potential as transformative OA treatments.

Coordination of Pickpocket ion channel delivery and dendrite growth in Drosophila sensory neurons.

Sensory neurons enable an organism to perceive external stimuli, which is essential for survival. The sensory capacity of a neuron depends on the elaboration of its dendritic arbor and the localization of sensory ion channels to the dendritic membrane. However, it is not well understood when and how ion channels localize to growing sensory dendrites and whether their delivery is coordinated with growth of the dendritic arbor. We investigated the localization of the DEG/ENaC/ASIC ion channel Pickpocket (Ppk) in the peripheral sensory neurons of developing fruit flies. We used CRISPR-Cas9 genome engineering approaches to tag endogenous Ppk1 and visualize it live, including monitoring Ppk1 membrane localization via a novel secreted split-GFP approach. Fluorescently tagged endogenous Ppk1 localizes to dendrites, as previously reported, and, unexpectedly, to axons and axon terminals. In dendrites, Ppk1 is present throughout actively growing dendrite branches and is stably integrated into the neuronal cell membrane during the expansive growth of the arbor. Although Ppk channels are dispensable for dendrite growth, we found that an over-active channel mutant severely reduces dendrite growth, likely by acting at an internal membrane and not the dendritic membrane. Our data reveal that the molecular motor dynein and recycling endosome GTPase Rab11 are needed for the proper trafficking of Ppk1 to dendrites. Based on our data, we propose that Ppk channel transport is coordinated with dendrite morphogenesis, which ensures proper ion channel density and distribution in sensory dendrites.

CRISPR-Driven Genome Engineering for Chorismate- and Anthranilate-Accumulating Corynebacterium Cell Factories.

In this study, we aimed to enhance the accumulation of chorismate (CHR) and anthranilate (ANT), key intermediates in the shikimate pathway, by modifying a shikimate over-producing recombinant strain of Corynebacterium glutamicum [19]. To achieve this, we utilized a CRISPR-driven genome engineering approach to compensate for the deletion of shikimate kinase (AroK) as well as ANT synthases (TrpEG) and ANT phosphoribosyltransferase (TrpD). In addition, we inhibited the CHR metabolic pathway to induce CHR accumulation. Further, to optimize the shikimate pathway, we overexpressed feedback inhibition-resistant Escherichia coli AroG and AroH genes, as well as C. glutamicum AroF and AroB genes. We also overexpressed QsuC and substituted shikimate dehydrogenase (AroE). In parallel, we optimized the carbon metabolism pathway by deleting the gntR family transcriptional regulator (IolR) and overexpressing polyphosphate/ATP-dependent glucokinase (PpgK) and glucose kinase (Glk). Moreover, acetate kinase (Ack) and phosphotransacetylase (Pta) were eliminated. Through our CRISPR-driven genome re-design approach, we successfully generated C. glutamicum cell factories capable of producing up to 0.48 g/L and 0.9 g/L of CHR and ANT in 1.3 mL miniature culture systems, respectively. These findings highlight the efficacy of our rational cell factory design strategy in C. glutamicum, which provides a robust platform technology for developing high-producing strains that synthesize valuable aromatic compounds, particularly those derived from the shikimate pathway metabolites.

A simple method to dramatically increase C. elegans germline microinjection efficiency

Genome manipulation methods in C. elegans require microinjecting DNA or ribonucleoprotein complexes into the microscopic core of the gonadal syncytium. These microinjections are technically demanding and represent a key bottleneck for all genome engineering and transgenic approaches in C. elegans. While there have been steady improvements in the ease and efficiency of genetic methods for C. elegans genome manipulation, there have not been comparable advances in the physical process of microinjection. Here, we report a simple and inexpensive method for handling worms using a paintbrush during the injection process that nearly tripled average microinjection rates compared to traditional worm handling methods. We found that the paintbrush increased injection throughput by substantially increasing both injection speeds and post-injection survival rates. In addition to dramatically and universally increasing injection efficiency for experienced personnel, the paintbrush method also significantly improved the abilities of novice investigators to perform key steps in the microinjection process. We expect that this method will benefit the C. elegans community by increasing the speed at which new strains can be generated and will also make microinjection-based approaches less challenging and more accessible to personnel and labs without extensive experience.

Two differentially stable rDNA loci coexist on the same chromosome and form a single nucleolus

The nucleolus is the most prominent membraneless compartment within the nucleus-dedicated to the metabolism of ribosomal RNA. Nucleoli are composed of hundreds of ribosomal DNA (rDNA) repeated genes that form large chromosomal clusters, whose high recombination rates can cause nucleolar dysfunction and promote genome instability. Intriguingly, the evolving architecture of eukaryotic genomes appears to have favored two strategic rDNA locations-where a single locus per chromosome is situated either near the centromere (CEN) or the telomere. Here, we deployed an innovative genome engineering approach to cut and paste to an ectopic chromosomal location-the ~1.5 mega-base rDNA locus in a single step using CRISPR technology. This "megablock" rDNA engineering was performed in a fused-karyotype strain of Saccharomyces cerevisiae. The strategic repositioning of this locus within the megachromosome allowed experimentally mimicking and monitoring the outcome of an rDNA migratory event, in which twin rDNA loci coexist on the same chromosomal arm. We showed that the twin-rDNA yeast readily adapts, exhibiting wild-type growth and maintaining rRNA homeostasis, and that the twin loci form a single nucleolus throughout the cell cycle. Unexpectedly, the size of each rDNA array appears to depend on its position relative to the CEN, in that the locus that is CEN-distal undergoes size reduction at a higher frequency compared to the CEN-proximal counterpart. Finally, we provided molecular evidence supporting a mechanism called paralogous cis-rDNA interference, which potentially explains why placing two identical repeated arrays on the same chromosome may negatively affect their function and structural stability.

Inhibition of Transforming Growth Factor-β Improves Primary Renal Tubule Cell Differentiation in Long-Term Culture.

Patient-oriented applications of cell culture include cell therapy of organ failure like chronic renal failure. Clinical deployment of a cell-based device for artificial renal replacement requires qualitative and quantitative fidelity of a cultured cell to its in vivo counterpart. Active specific apicobasal ion transport reabsorbs 90-99% of the filtered load of salt and water in the kidney. In a bioengineered kidney, tubular transport concentrates wastes and eliminates the need for hemodialysis, but renal tubule cells in culture transport little or no salt and water due to dedifferentiation that mammalian cells undergo in vitro thereby losing important cell-type specific functions. We previously identified transforming growth factor-β (TGF-β) as a signaling pathway necessary for in vitro differentiation of renal tubule cells. Inhibition of TGF-β receptor-1 led to active and inhibitable electrolyte and water transport by primary human renal tubule epithelial cells in vitro. Addition of metformin increased transport, in the context of a transient effect on 5'-AMP-activated kinase phosphorylation. These data motivated us to examine whether increased transport was an idiosyncratic effect of SB431542, probe pathways downstream of TGF-β receptors possibly responsible for the improved differentiation, evaluate whether TGF-β inhibition induced a range of differentiated tubule functions, and to explore crosstalk between the effects of SB431542 and metformin. In this study, we use multiple small-molecule inhibitors of canonical and noncanonical pathways to confirm that inhibition of canonical TGF-β signaling caused the increased apicobasal transport. Hallmarks of proximal tubule cell function, including sodium reabsorption, para-amino hippurate excretion, and glucose uptake increased with TGF-β inhibition, and the specificity of the response was shown using inhibitors of each transport protein. We did not find any evidence of crosstalk between metformin and SB431542. These data suggest that the TGF-β signaling pathway governs multiple features of differentiation in renal proximal tubule cells in vitro. Inhibition of TGF-β by pharmacologic or genome engineering approaches may be a viable approach to enhancing differentiated function of tubule cells in vitro. Impact statement Cell therapy of renal failure requires qualitative and quantitative fidelity between in vitro and in vivo phenotypes, which has been elusive. We show that control of transforming growth factor-β signaling can promote differentiation of renal tubule cells grown in artificial environments. This is a key enabling step for cell therapy of renal failure.

Application of CRISPR-Cas9 Gene Editing for HIV Host Factor Discovery and Validation.

Human Immunodeficiency Virus (HIV) interacts with a wide array of host factors at each stage of its lifecycle to facilitate replication and circumvent the immune response. Identification and characterization of these host factors is critical for elucidating the mechanism of viral replication and for developing next-generation HIV-1 therapeutic and curative strategies. Recent advances in CRISPR-Cas9-based genome engineering approaches have provided researchers with an assortment of new, valuable tools for host factor discovery and interrogation. Genome-wide screening in a variety of in vitro cell models has helped define the critical host factors that play a role in various cellular and biological contexts. Targeted manipulation of specific host factors by CRISPR-Cas9-mediated gene knock-out, overexpression, and/or directed repair have furthermore allowed for target validation in primary cell models and mechanistic inquiry through hypothesis-based testing. In this review, we summarize several CRISPR-based screening strategies for the identification of HIV-1 host factors and highlight how CRISPR-Cas9 approaches have been used to elucidate the molecular mechanisms of viral replication and host response. Finally, we examine promising new technologies in the CRISPR field and how these may be applied to address critical questions in HIV-1 biology going forward.

Genome Engineering for Next-Generation Cellular Immunotherapies.

Over the past decade, cellular immunotherapies such as CAR-T, TCR-T, and NK cell therapies have achieved tremendous success in cancer treatment. However, various challenges and obstacles remain, including antigen escape, immunosuppression in the tumor microenvironment, toxicities, and on-target off-tumor effects. Recent strategies for overcoming these roadblocks have included the use of genome engineering. Multiplexed CRISPR-Cas and synthetic biology approaches facilitate the development of cell therapies with higher potency and sophisticated modular control; they also offer a toolkit for allogeneic therapy development. Engineering approaches have targeted genetic modifications to enhance long-term persistence through cytokine modulation, knockout of genes mediating immunosuppressive signals, and genes such as the endogenous TCR and MHC-I that elicit adverse host-graft interactions in an allogeneic context. Genome engineering approaches for other immune cell types are also being explored, such as CAR macrophages and CAR-NK cells. Future therapeutic development of cellular immunotherapies may also be guided by novel target discovery through unbiased CRISPR genetic screening approaches.

TET1 regulates gene expression and repression of endogenous retroviruses independent of DNA demethylation.

DNA methylation (5-methylcytosine (5mC)) is critical for genome stability and transcriptional regulation in mammals. The discovery that ten-eleven translocation (TET) proteins catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) revolutionized our perspective on the complexity and regulation of DNA modifications. However, to what extent the regulatory functions of TET1 can be attributed to its catalytic activity remains unclear. Here, we use genome engineering and quantitative multi-omics approaches to dissect the precise catalytic vs. non-catalytic functions of TET1 in murine embryonic stem cells (mESCs). Our study identifies TET1 as an essential interaction hub for multiple chromatin modifying complexes and a global regulator of histone modifications. Strikingly, we find that the majority of transcriptional regulation depends on non-catalytic functions of TET1. In particular, we show that TET1 is critical for the establishment of H3K9me3 and H4K20me3 at endogenous retroviral elements (ERVs) and their silencing that is independent of its canonical role in DNA demethylation. Furthermore, we provide evidence that this repression of ERVs depends on the interaction between TET1 and SIN3A. In summary, we demonstrate that the non-catalytic functions of TET1 are critical for regulation of gene expression and the silencing of endogenous retroviruses in mESCs.

Genomic and epigenetic landscapes drive CRISPR-based genome editing in Bifidobacterium

Bifidobacterium is a commensal bacterial genus ubiquitous in the human gastrointestinal tract, which is associated with a range of health benefits. The advent of CRISPR-based genome editing technologies provides opportunities to investigate the genetics of important bacteria and transcend the lack of genetic tools in bifidobacteria to study the basis for their health-promoting attributes. Here, we repurpose the endogenous type I-G CRISPR-Cas system and adopt an exogenous CRISPR base editor for genome engineering in B. animalis subsp. lactis, demonstrating that both genomic and epigenetic contexts drive editing outcomes across strains. We reprogrammed the endogenous type I-G system to screen for naturally occurring large deletions up to 27 kb and to generate a 500-bp deletion in tetW to abolish tetracycline resistance. A CRISPR-cytosine base editor was optimized to install C•G-to-T•A amber mutations to resensitize multiple B. lactis strains to tetracycline. Remarkably, we uncovered epigenetic patterns that are distributed unevenly among B. lactis strains, despite their genomic homogeneity, that may contribute to editing efficiency variability. Insights were also expanded to Bifidobacterium longum subsp. infantis to emphasize the broad relevance of these findings. This study highlights the need to develop individualized CRISPR-based genome engineering approaches for distinct bacterial strains and opens avenues for engineering of next generation probiotics.

Abstract LE03-01: Functional and computational approaches to uncover selection advantages of cancer aneuploidy

Abstract Aneuploidy, including the gain or loss of whole chromosomes or chromosome arms, is a near-universal feature of cancer. We previously applied methods that define chromosome arm aneuploidy to over 10,000 tumors in the Cancer Genome Atlas (TCGA). Cancer subtypes are often characterized by tumor specific patterns of chromosome arm copy number alterations and breakpoints; for example, squamous cell carcinomas (SCCs) from different tissues of origin are characterized by chromosome 3p (chr3p) loss and chromosome 3q (chr3q) gain. From the TCGA aneuploidy data, we developed an algorithm called BISCUT to distinguish peak regions of aneuploidy breakpoints on each chromosome arm. BISCUT identified loci affected by broad copy number alterations that provide fitness advantages or disadvantages both within individual cancer types and across cancers. Our analyses are consistent with selection being the primary driver of aneuploidy events in cancer. Recent advances in genome engineering allow generation of large chromosomal alterations and validation of findings from patient genomic data. We used the CRISPR-Cas9 system to delete one copy of chr3p in a human immortalized lung epithelial cell line similar to the putative cell-of-origin in lung SCC. Consistent with patient data, expression of chr3p genes was decreased upon deletion, as well as increased expression of interferon response genes. Cells with chr3p deletion initially proliferated more slowly than their siblings. Interestingly, after several passages in culture, this proliferation defect was rescued in chr3p deleted cells. Genome sequencing and karyotype analyses suggested that this was partially the result of chr3 duplication, with cells transitioning to a state of chr3q gain. Isolated cells with chr3q gain show deficiencies in squamous differentiation in organoid culture. Our genome engineering approach to model chromosome arm aneuploidies provides a robust model that will address a gap in our understanding of aneuploidy in cancer. Citation Format: Alison Marie Taylor. Functional and computational approaches to uncover selection advantages of cancer aneuploidy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LE03-01.

Recent advancements in CRISPR/Cas technology for accelerated crop improvement.

Precise genome engineering approaches could be perceived as a second paradigm for targeted trait improvement in crop plants, with the potential to overcome the constraints imposed by conventional CRISPR/Cas technology. The likelihood of reduced agricultural production due to highly turbulent climatic conditions increases as the global population expands. The second paradigm of stress-resilient crops with enhanced tolerance and increased productivity against various stresses is paramount to supportglobal production and consumption equilibrium. Although traditional breeding approaches have substantially increased crop production and yield, effective strategies are anticipated to restore crop productivity even further in meeting the world's increasing food demands. CRISPR/Cas, which originated in prokaryotes, has surfaced as a coveted genome editing tool in recent decades, reshaping plant molecular biology in unprecedented ways and paving the way for engineering stress-tolerant crops. CRISPR/Cas is distinguished by its efficiency, high target specificity, and modularity, enables precise genetic modification of crop plants, allowing for the creation of allelic variations in the germplasm and the development of novel and more productive agricultural practices. Additionally, a slew of advanced biotechnologies premised on the CRISPR/Cas methodologies have augmented fundamental research and plant synthetic biology toolkits. Here, we describe gene editing tools, including CRISPR/Cas and its imitative tools, such as base and prime editing, multiplex genome editing, chromosome engineering followed by their implications in crop genetic improvement. Further, we comprehensively discuss the latest developments of CRISPR/Cas technology including CRISPR-mediated gene drive, tissue-specific genome editing, dCas9 mediated epigenetic modification and programmed self-elimination of transgenes in plants. Finally, we highlight the applicability and scope of advanced CRISPR-based techniques in crop genetic improvement.

Feeder Cells at the Interface of Natural Killer Cell Activation, Expansion and Gene Editing.

Genome engineered natural killer (NK) cell therapies are emerging as a promising cancer immunotherapy platform with potential advantages and remaining uncertainties. Feeder cells induce activation and proliferation of NK cells via cell surface receptor-ligand interactions, supported by cytokines. Feeder cell expanded NK cell products have supported several NK cell adoptive transfer clinical trials over the past decade. Genome engineered NK cell therapies, including CAR-NK cells, seek to combine innate and alloreactive NK cell anti-tumor activity with antigen specific targeting or additional modifications aimed at improving NK cell persistence, homing or effector function. The profound activating and expansion stimulus provided by feeder cells is integral to current applications of clinical-scale genome engineering approaches in donor-derived, primary NK cells. Herein we explore the complex interactions that exist between feeder cells and both viral and emerging non-viral genome editing technologies in NK cell engineering. We focus on two established clinical-grade feeder systems; Epstein-Barr virus transformed lymphoblastoid cell lines and genetically engineered K562.mbIL21.4-1BBL feeder cells.

Prime editing efficiency and fidelity are enhanced in the absence of mismatch repair

Prime editing (PE) is a powerful genome engineering approach that enables the introduction of base substitutions, insertions and deletions into any given genomic locus. However, the efficiency of PE varies widely and depends not only on the genomic region targeted, but also on the genetic background of the edited cell. Here, to determine which cellular factors affect PE efficiency, we carry out a focused genetic screen targeting 32 DNA repair factors, spanning all reported repair pathways. We show that, depending on cell line and type of edit, ablation of mismatch repair (MMR) affords a 2–17 fold increase in PE efficiency, across several human cell lines, types of edits and genomic loci. The accumulation of the key MMR factors MLH1 and MSH2 at PE sites argues for direct involvement of MMR in PE control. Our results shed new light on the mechanism of PE and suggest how its efficiency might be optimised.

Improvement of base editors and prime editors advances precision genome engineering in plants.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas)-mediated gene disruption has revolutionized biomedical research as well as plant and animal breeding. However, most disease-causing mutations and agronomically important genetic variations are single base polymorphisms (single-nucleotide polymorphisms) that require precision genome editing tools for correction of the sequences. Although homology-directed repair of double-stranded breaks (DSBs) can introduce precise changes, such repairs are inefficient in differentiated animal and plant cells. Base editing and prime editing are two recently developed genome engineering approaches that can efficiently introduce precise edits into target sites without requirement of DSB formation or donor DNA templates. They have been applied in several plant species with promising results. Here, we review the extensive literature on improving the efficiency, target scope, and specificity of base editors and prime editors in plants. We also highlight recent progress on base editing in plant organellar genomes and discuss how these precision genome editing tools are advancing basic plant research and crop breeding.

209.7: An Inducible Genome Engineering Approach for Preventing Immune Rejection of hESC-derived Beta Cells

209.7: An Inducible Genome Engineering Approach for Preventing Immune Rejection of hESC-derived Beta Cells