Oligonucleotide Templates Research Articles

Pythia: Non-random DNA repair allows predictable CRISPR/Cas9 integration and gene editing.

CRISPR-based genome engineering holds enormous promise for basic science and therapeutic applications. Integrating and editing DNA sequences is still challenging in many cellular contexts, largely due to insufficient control of the repair process. We find that repair at the genome-cargo interface is predictable by deep-learning models and adheres to sequence context specific rules. Based on in silico predictions, we devised a strategy of triplet base-pair repeat repair arms that correspond to microhomologies at double-strand breaks (trimologies), which facilitated integration of large cargo (>2 kb) and protected the targeted locus and transgene from excessive damage. Successful integrations occurred in >30 loci in human cells and in in vivo models. Germline transmissible transgene integration in Xenopus , and endogenous tagging of tubulin in adult mice brains demonstrated integration during early embryonic cleavage and in non-dividing differentiated cells. Further, optimal repair arms for single- or double nucleotide edits were predictable, and facilitated small edits in vitro and in vivo using oligonucleotide templates. We provide a design-tool (Pythia, pythia-editing.org ) to optimize custom integration, tagging or editing strategies. Pythia will facilitate genomic integration and editing for experimental and therapeutic purposes for a wider range of target cell types and applications.

The influence of nucleotide context on non-specific amplification of DNA with Bst exo- DNA polymerase

In recent years, nucleic acid amplification methods that proceed in isothermal mode and require the use of DNA polymerases with strand-displacement activity have become increasingly widespread. Among these, the most popular is the Bst exo- polymerase, but it tends to carry out nonspecific DNA synthesis through multimerization. This study shows the influence of the nucleotide sequence on the binding of Bst exo- with DNA and on the efficiency of multimerization initiation. On single-stranded trinucleotides (sst) and dinucleotide duplexes (dst), molecular docking revealed the preference for binding of the “closed” form of Bst exo- to purine-rich sequences, especially those containing dG at the 3′ end of the synthesized strand. The data obtainedin silicowere confirmed in experiments using oligonucleotide templates that differ in the structure of the 3′- and 5′-terminal motifs. It has been shown that templates with an oligopurine 3′-terminal fragment and an oligopyrimidine 5′-terminal part contribute to an earlier start of multimerization. The obtained data can be used at the stage of selecting optimal nucleotide sequences for isothermal amplification, ensuring more reliable results. Thus, to avoid multimerization, DNA templates and primers containing terminal dA and/or dG nucleotides should be excluded.

Influence of Nucleotide Context on Non-Specific Amplification of DNA with Bst exo- DNA Polymerase.

Isothermal nucleic acids amplification that requires DNA polymerases with strand-displacement activity gained more attention in the last two decades. Among the DNA polymerases with strand-displacement activity, Bst exo- is the most widely used. However, it tends to carry out nonspecific DNA synthesis through multimerization. In this study, the effect of nucleotide sequence on the Bst exo- binding with DNA and on the efficiency of multimerization initiation, are reported. Preference for binding of the "closed" form of Bst exo- to the purine-rich DNA sequences, especially those containing dG at the 3'-end of the growing chain was revealed using molecular docking of the single-stranded trinucleotides (sst) and trinucleotide duplexes (dst). The data obtained in silico were confirmed in the experiments using oligonucleotide templates that differ in the structure of the 3'- and 5'-terminal motifs. It has been shown that templates with the oligopurine 3'-terminal fragment and oligopyrimidine 5'-terminal part contribute to the earlier start of multimerization. The results can be used for design of nucleotide sequences suitable for reliable isothermal amplification. To avoid multimerization, DNA templates and primers containing terminal dA and/or dG nucleotides should be excluded.

Highly efficient in vivo C-to-T base editing in Atlantic salmon (Salmo salar) – A step towards aquaculture precision breeding

The salmon industry has the potential to provide nutritious food to the expanding world population. However, the industry faces several sustainability challenges related to environmental impact and fish welfare. Genome editing presents a promising approach for precision breeding, where small, targeted changes to the fish genome may facilitate the introduction of advantageous traits like disease resistance, delayed maturation, or enhanced growth. This study presents the first application of base editing in an aquaculture fish species. By co-injecting cytidine base editor AncBE4max mRNA and gRNA targeting the pigmentation gene solute carrier family 45 member 2 in Atlantic salmon embryos, we were able to induce a specific C-to-T conversion, thereby introducing a premature stop codon and achieving an albino phenotype. High-throughput sequencing revealed an average of 50–66% correct conversion of the target base depending on the base editor mRNA concentration and up to 89% in a single individual. Our results suggest that AncBE4max provides a simple and efficient approach to making precise single nucleotide edits in the Atlantic salmon genome. Additionally, we used conventional CRISPR/Cas9 combined with oligonucleotide templates to insert point mutations at various positions up- and downstream of the Cas9 cleavage site, demonstrating that the insertion efficiency was affected by the position of the mutation. The application of genome editing tools that allow for precise changes in this species is a step towards aquaculture precision breeding and the introduction of traits promoting a more sustainable industry.

Quantification of all 12 canonical ribonucleotides by real-time fluorogenic in vitro transcription.

Enzymatic methods to quantify deoxyribonucleoside triphosphates have existed for decades. In contrast, no general enzymatic method to quantify ribonucleoside triphosphates (rNTPs), which drive almost all cellular processes and serve as precursors of RNA, exists to date. ATP can be measured with an enzymatic luminometric method employing firefly luciferase, but the quantification of other ribonucleoside mono-, di-, and triphosphates is still a challenge for a non-specialized laboratory and practically impossible without chromatography equipment. To allow feasible quantification of ribonucleoside phosphates in any laboratory with typical molecular biology and biochemistry tools, we developed a robust microplate assay based on real-time detection of the Broccoli RNA aptamer during in vitro transcription. The assay employs the bacteriophage T7 and SP6 RNA polymerases, two oligonucleotide templates encoding the 49-nucleotide Broccoli aptamer, and a high-affinity fluorogenic aptamer-binding dye to quantify each of the four canonical rNTPs. The inclusion of nucleoside mono- and diphosphate kinases in the assay reactions enabled the quantification of the mono- and diphosphate counterparts. The assay is inherently specific and tolerates concentrated tissue and cell extracts. In summary, we describe the first chromatography-free method to quantify ATP, ADP, AMP, GTP, GDP, GMP, UTP, UDP, UMP, CTP, CDPand CMP in biological samples.

Fluorescent PCR-based Screening Methods for Precise Knock-in of Small DNA Fragments and Point Mutations in Zebrafish.

Generation of zebrafish (Danio rerio) models with targeted insertion of epitope tags and point mutations is highly desirable for functional genomics and disease modeling studies. Currently, CRISPR/Cas9-mediated knock-in is the method of choice for insertion of exogeneous sequences by providing a repair template for homology-directed repair (HDR). A major hurdle in generating knock-in models is the labor and cost involved in screening of injected fish to identify the precise knock-in events due to low efficiency of the HDR pathway in zebrafish. Thus, we developed fluorescent PCR-based high-throughput screening methods for precise knock-in of epitope tags and point mutations in zebrafish. Here, we provide a step-by-step guide that describes selection of an active sgRNA near the intended knock-in site, design of single-stranded oligonucleotide (ssODN) templates for HDR, quick validation of somatic knock-in using injected embryos, and screening for germline transmission of precise knock-in events to establish stable lines. Our screening method relies on the size-based separation of all fragments in an amplicon by fluorescent PCR and capillary electrophoresis, thus providing a robust and cost-effective strategy. Although we present the use of this protocol for insertion of epitope tags and point mutations, it can be used for insertion of any small DNA fragments (e.g., LoxP sites, in-frame codons). Furthermore, the screening strategy described here can be used to screen for precise knock-in of small DNA sequences in any model system, as PCR amplification of the target region is its only requirement. Key features This protocol expands the use of fluorescent PCR and CRISPR-STAT for screening of precise knock-in of small insertions and point mutations in zebrafish. Allows validation of selected sgRNA and HDR template within two weeks by somatic knock-in screening. Allows robust screening of point mutations by combining restriction digest with CRISPR-STAT. Graphical overview Overview of the three-phase knock-in pipeline in zebrafish (created with BioRender.com).

PepSeq: a fully in vitro platform for highly multiplexed serology using customizable DNA-barcoded peptide libraries.

PepSeq is an in vitro platform for building and conducting highly multiplexed proteomic assays against customizable targets by using DNA-barcoded peptides. Starting with a pool of DNA oligonucleotides encoding peptides of interest, this protocol outlines a fully in vitro and massively parallel procedure for synthesizing the encoded peptides and covalently linking each to a corresponding cDNA tag. The resulting libraries of peptide/DNA conjugates can be used for highly multiplexed assays that leverage high-throughput sequencing to profile the binding or enzymatic specificities of proteins of interest. Here, we describe the implementation of PepSeq for fast and cost-effective epitope-level analysis of antibody reactivity across hundreds of thousands of peptides from <1 µl of serum or plasma input. This protocol includes the design of the DNA oligonucleotide library, synthesis of DNA-barcoded peptide constructs, binding of constructs to sample, preparation for sequencing and data analysis. Implemented in this way, PepSeq can be used for a number of applications, including fine-scale mapping of antibody epitopes and determining a subject's pathogen exposure history. The protocol is divided into two main sections: (i) design and synthesis of DNA-barcoded peptide libraries and (ii) use of libraries for highly multiplexed serology. Once oligonucleotide templates are in hand, library synthesis takes 1-2 weeks and can provide enough material for hundreds to thousands of assays. Serological assays can be conducted in 96-well plates and generate sequencing data within a further ~4 d. A suite of software tools, including the PepSIRF package, are made available to facilitate the design of PepSeq libraries and analysis of assay data.

The most common disease-causing mutation of factor XIII deficiency is corrected by CRISPR/CAS9 gene editing system.

Factor XIII (FXIII) deficiency is one of the most severe congenital bleeding disorders, with an estimated incidence of one person per one million. Patients with severe FXIII deficiency present a wide range of clinical manifestations, including umbilical cord bleeding, intracranial haemorrhage and recurrent miscarriages. Due to the high rate of life-threatening bleeding, primary prophylaxis is mandatory from the time of diagnosis. Although replacement therapy is the most common therapeutic choice, gene therapy remains the only curative option. In the present study, we assessed the efficacy of the clustered regularly interspaced short palindromic repeats - CRISPR-associated protein 9 (CRISPR/Cas9) system in the correction of the most common FXIII disease-causing mutation (c.562 T > C). A dermal fibroblast was harvested from the human skin biopsy of a young patient with FXIII deficiency. Sanger sequencing was used to confirm the presence of c.562 T>C mutation in the patient and in the harvested fibroblasts. PX459 vector was digested with BbsI restriction enzyme, and after annealing and ligation of two 20-bp guide-RNAs (g-RNAs) close to the PAM (NGG) sequence, the constructed vectors were amplified in Escherichia coli Top 10. Transfection was performed by a nucleofector device, and DNA extraction was performed after puromycin selection and serial dilution from potentially transfected colonies. A 50-bp template oligonucleotide was used to aid homologous repair for correction of the underlying mutation and synonymous mutation as an internal control. The synonymous mutation (AAT to ACT) near the mutation site was used as internal control. Sanger sequencing was done in order to check the gene correction. The c.562 T > C mutation was detected in homozygote state in the primary fibroblasts of the patient and wild-type alleles were confirmed in the normal individual. Colony PCR and sequencing revealed successful cloning of the designed gRNAs. The detected mutation was corrected from a homozygote mutant state (c.562 T > C) to a homozygote wild type in transfected dermal fibroblasts of the patient. The control mutation, as an internal control, was also corrected in the same fibroblasts in the heterozygote manner. The result of the study shows that the CRISPR/CAS9 gene editing system is an effective tool for correction of point mutations in transfected fibroblasts of patients with congenital FXIII deficiency and represents a new, potentially curative, option.

A portable system for isothermal amplification and detection of exosomal microRNAs

Exosomal microRNAs (miRNAs) play a key role in cell-cell communication to regulate gene expression in target cells and have great potential as biomarkers for disease diagnosis. This paper reports an on-chip exosomal miRNA amplification and detection system for rapid analysis of exosomal miRNAs. The compact system consists of two connected flow cells for processing exosomes and detecting miRNAs, respectively. The miRNAs extracted from exosomes were quantitatively measured using the on-chip exponential amplification reaction (EXPAR) assay. The sensor chip was designed to store multiple oligonucleotide templates for the EXPAR, mix sample and reagent, and simultaneously analyze multiple exosomal miRNAs of interest. To facilitate the miRNA analysis, a portable detection instrument was built on an IoT platform using a low-cost microcontroller to execute the EXPAR assay, collect fluorescent images, and analyze amplification curves. Here, we studied the miRNA profiles carried by exosomes derived from three different phenotypes of tissue macrophages. The affordable instrument, rapid assay, multiplexed analysis, as well as disposable sensor chip, would boost the development of point-of-care liquid biopsy tests using exosomal miRNAs.

Labeling of Synthetic Oligonucleotides Using the Klenow Fragment of E. coli DNA Polymerase I.

In this method, a short primer is hybridized to an oligonucleotide template whose sequence is the complement of the desired radiolabeled probe. The primer is then extended using the Klenow fragment to incorporate [α-32P]dNTPs in a template-directed manner. After the reaction, the template and product are separated by denaturation followed by electrophoresis through a polyacrylamide gel under denaturing conditions. With this method, it is possible to generate oligonucleotide probes that contain several radioactive atoms per molecule of oligonucleotide and to achieve specific activities as high as 2 × 1010 cpm/µg of probe. Because the end product of the reaction is dsDNA, whose strands must be separated and the labeled product isolated, this method is generally not used to prepare nonradiolabeled oligonucleotides.

A Consensus Model of Homology-Directed Repair Initiated by CRISPR/Cas Activity.

The mechanism of action of ssODN-directed gene editing has been a topic of discussion within the field of CRISPR gene editing since its inception. Multiple comparable, but distinct, pathways have been discovered for DNA repair both with and without a repair template oligonucleotide. We have previously described the ExACT pathway for oligo-driven DNA repair, which consisted of a two-step DNA synthesis-driven repair catalyzed by the simultaneous binding of the repair oligonucleotide (ssODN) upstream and downstream of the double-strand break. In order to better elucidate the mechanism of ExACT-based repair, we have challenged the assumptions of the pathway with those outlines in other similar non-ssODN-based DNA repair mechanisms. This more comprehensive iteration of the ExACT pathway better described the many different ways where DNA repair can occur in the presence of a repair oligonucleotide after CRISPR cleavage, as well as how these previously distinct pathways can overlap and lead to even more unique repair outcomes.

The Emergence of RNA from the Heterogeneous Products of Prebiotic Nucleotide Synthesis.

Recent advances in prebiotic chemistry are beginning to outline plausible pathways for the synthesis of the canonical ribonucleotides and their assembly into oligoribonucleotides. However, these reaction pathways suggest that many noncanonical nucleotides are likely to have been generated alongside the standard ribonucleotides. Thus, the oligomerization of prebiotically synthesized nucleotides is likely to have led to a highly heterogeneous collection of oligonucleotides comprised of a wide range of types of nucleotides connected by a variety of backbone linkages. How then did relatively homogeneous RNA emerge from this primordial heterogeneity? Here we focus on nonenzymatic template-directed primer extension as a process that would have strongly enriched for homogeneous RNA over the course of multiple cycles of replication. We review the effects on copying the kinetics of nucleotides with altered nucleobase and sugar moieties, when they are present as activated monomers and when they are incorporated into primer and template oligonucleotides. We also discuss three variations in backbone connectivity, all of which are nonheritable and regenerate native RNA upon being copied. The kinetic superiority of RNA synthesis suggests that nonenzymatic copying served as a chemical selection mechanism that allowed relatively homogeneous RNA to emerge from a complex mixture of prebiotically synthesized nucleotides and oligonucleotides.

Generating Mutant Renal Cell Lines Using CRISPR Technologies.

Gene editing using the CRISPR/Cas9 system is an extremely efficient approach for generating mutations within the genomic DNA of immortalized cell lines. This procedure begins with a straightforward cloning step to generate a single plasmid encoding the Cas9 enzyme as well as a synthetic guide RNA (sgRNA) which is selected to target specific sites within the genome. This plasmid is transfected into cells either alone, in order to generate random insertion-deletion alleles ("indels") at the desired locus via the nonhomologous end-joining pathway, or in conjunction with a homology-directed repair template oligonucleotide to generate a specific point mutation. Here we describe a procedure to perform gene editing in IMCD3 and HEK293 cells and to subsequently isolate clonal cell lines carrying mutations of interest.

Generation of deletions and precise point mutations in Dictyostelium discoideum using the CRISPR nickase.

The CRISPR/Cas9 system enables targeted genome modifications across a range of eukaryotes. Although we have reported that transient introduction of all-in-one vectors that express both Cas9 and sgRNAs can efficiently induce multiple gene knockouts in Dictyostelium discoideum, concerns remain about off-target effects and false-positive amplification during mutation detection via PCR. To minimise these effects, we modified the system to permit gene deletions of greater than 1 kb via use of paired sgRNAs and Cas9 nickase. An all-in-one vector expressing the Cas9 nickase and sgRNAs was transiently introduced into D. discoideum, and the resulting mutants showed long deletions with a relatively high efficiency of 10–30%. By further improving the vector, a new dual sgRNA expression vector was also constructed to allow simultaneous insertion of two sgRNAs via one-step cloning. By applying this system, precise point mutations and genomic deletions were generated in the target locus via simultaneous introduction of the vector and a single-stranded oligonucleotide template without integrating a drug resistance cassette. These systems enable simple and straightforward genome editing that requires high specificity, and they can serve as an alternative to the conventional homologous recombination-based gene disruption method in D. discoideum.

Transient up-regulation of miR-155-3p by lipopolysaccharide in primary human monocyte-derived macrophages results in RISC incorporation but does not alter TNF expression.

Background: The innate immune response is a tightly regulated process that reacts rapidly in response to pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS). Evidence is accumulating that microRNAs contribute to this, although few studies have examined the early events that constitute the "primary" response. Methods: LPS-dependent changes to miRNA expression were studied in primary human monocyte-derived macrophages (1°MDMs). An unbiased screen by microarray was validated by qPCR and a method for the absolute quantitation of miRNAs was also developed, utilising 5' phosphorylated RNA oligonucleotide templates. RNA immunoprecipitation was performed to explore incorporation of miRNAs into the RNA-induced silencing complex (RISC). The effect of miRNA functional inhibition on TNF expression (mRNA and secretion) was investigated. Results: Of the 197 miRNAs expressed in 1°MDMs, only five were induced >1.5-fold. The most strongly induced was miR-155-3p, the partner strand to miR-155-5p, which are both derived from the MIR155HG/BIC gene (pri-miR-155). The abundance of miR-155-3p was induced transiently ~250-fold at 2-4hrs and then returned towards baseline, mirroring pri-miR-155. Other PAMPs, IL-1β, and TNF caused similar responses. IL-10, NF-κB, and JNK inhibition reduced these responses, unlikecytokine-suppressing mycolactone. Absolute quantitation revealed that miRNA abundance varies widely from donor-to-donor, and showed that miR-155-3p abundance is substantially less than miR-155-5p in unstimulated cells. However, at its peak there were 446-1,113 copies/cell, and miR-155-3p was incorporated into the RISC with an efficiency similar to miR-16-5p and miR-155-5p. Inhibition of neither miRNA affected TNF secretion after 2hrs in 1°MDMs, but technical challenges here are noted. Conclusions: Dynamic regulation of miRNAs during the primary response is rare, with the exception of miR-155-3p. Further work is required to establish whether its low abundance, even at the transient peak, is sufficient for biological activity and to determine whether there are specific mechanisms determining its biogenesis from miR-155 precursors.

Transient cotransformation of CRISPR/Cas9 and oligonucleotide templates enables efficient editing of target loci in Physcomitrella patens.

Abstract Recent years, the bryophyte moss Physcomitrella patens has become an emerging model organism for studying conserved signaling pathways and developmental processes during plant evolution. Its short life cycle, ease of cultivation, and high rate of homologous recombination have made it an ideal system for genetic analysis. However, the presence of highly redundant genes and the difficulty of isolating hypomorphic mutants have limited its broader use. Here we developed a simple, fast, and efficient method to generate customized mutants in P. patens. We show that transient cotransformation of CRISPR/Cas9 and oligonucleotide templates enables microindel knock-in with high efficiency and accuracy. Using this method, we generated strains carrying various types of mutations, including amino acid substitution, out-of-frame deletion/insertion, splice site alteration, and small tag integration. We also demonstrate that multiplex gene editing can be efficiently achieved to generate putative null and hypomorphic mutants of redundant genes in one step. Thus our method will not only simplify multiple-gene knockout, but also allows the generation of hypomorphic mutants of genes of interest, especially those that are essential for viability.

Transient up-regulation of miR-155-3p by lipopolysaccharide in primary human monocyte-derived macrophages results in RISC incorporation but does not alter TNF expression

Background: The innate immune response is a tightly regulated process that reacts rapidly in response to pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS). Evidence is accumulating that microRNAs contribute to this, although few studies have examined the early events that constitute the “primary” response. Methods: LPS-dependent changes to miRNA expression were studied in primary human monocyte-derived macrophages (1°MDMs). An unbiased screen by microarray was validated by qPCR and a method for the absolute quantitation of miRNAs was also developed, utilising 5’ phosphorylated RNA oligonucleotide templates. RNA immunoprecipitation was performed to explore incorporation of miRNAs into the RNA-induced silencing complex (RISC). The effect of miRNA functional inhibition on TNF expression (mRNA and secretion) was investigated. Results: Of the 197 miRNAs expressed in 1°MDMs, only five were induced >1.5-fold. The most strongly induced was miR-155-3p, the partner strand to miR-155-5p, which are both derived from the MIR155HG/BIC gene (pri-miR-155). The abundance of miR-155-3p was induced transiently ~250-fold at 2-4hrs and then returned towards baseline, mirroring pri-miR-155. Other PAMPs, IL-1β, and TNF caused similar responses. IL-10, NF-κB, and JNK inhibition reduced these responses, unlike cytokine-suppressing mycolactone. Absolute quantitation revealed that miRNA abundance varies widely from donor-to-donor, and showed that miR-155-3p abundance is substantially less than miR-155-5p in unstimulated cells. However, at its peak there were 446-1,113 copies/cell, and miR-155-3p was incorporated into the RISC with an efficiency similar to miR-16-5p and miR-155-5p. Inhibition of neither miRNA affected TNF secretion after 2hrs in 1°MDMs, but technical challenges here are noted. Conclusions: Dynamic regulation of miRNAs during the primary response is rare, with the exception of miR-155-3p. Further work is required to establish whether its low abundance, even at the transient peak, is sufficient for biological activity and to determine whether there are specific mechanisms determining its biogenesis from miR-155 precursors

Methods for Correction of the Single-Nucleotide Substitution c.840C>T in Exon 7 of the SMN2 Gene.

The CRISPR/Cas technology has a great potential in the treatment of many hereditary diseases. One of the prospective models for the CRISPR/Cas-mediated therapy is spinal muscular atrophy (SMA), a disease caused by deletion of the SMN1 gene that encodes the SMN protein required for the survival of motor neurons. SMA patients' genomes contain either single or several copies of SMN2 gene, which is a paralog of SMN1. Exon 7 of SMN2 has the single-nucleotide substitution c.840C>T leading to the defective splicing and decrease in the amounts of the full-length SMN. The objective of this study was to create and test gene-editing systems for correction of the single-nucleotide substitution c.840C>T in exon 7 of the SMN2 gene in fibroblasts, induced pluripotent stem cells, and motor neuron progenitors derived from a SMA patient. For this purpose, we used plasmid vectors expressing CRISPR/Cas9 and CRISPR/Cpf1, plasmid donor, and 90-nt single-stranded oligonucleotide templates that were delivered to the target cells by electroporation. Although sgRNA_T2 and sgRNA_T3 guiding RNAs were more efficient than sgRNA_T1 in fibroblasts (p < 0.05), no significant differences in the editing efficiency of sgRNA_T1, sgRNA_T2, and sgRNA_T3 was observed in patient-specific induced pluripotent stem cells and motor neuron progenitors. The highest editing efficiency in induced pluripotent stem cells and motor neuron progenitors was demonstrated by the sgRNA_T1 and 90-nt single-stranded oligonucleotide donors.

Nonenzymatic Template-Directed Synthesis of Mixed-Sequence 3′-NP-DNA up to 25 Nucleotides Long Inside Model Protocells

Efficiently copying mixed-sequence oligonucleotide templates nonenzymatically is a long-standing problem both with respect to the origin of life, and with regard to bottom up efforts to synthesize artificial living systems. Here we report an efficient and sequence-general nonenzymatic process in which RNA templates direct the synthesis of a complementary strand composed of N3′→P5′ phosphoramidate DNA (3′-NP-DNA) using 3′-amino-2′,3′-dideoxyribonucleotides activated with 2-aminoimidazole. Using only the four canonical nucleobases (A, G, C, and T) of modern DNA, we demonstrate the chemical copying of a variety of mixed-sequence RNA templates, both in solution and within model protocells, into complementary 3′-NP-DNA strands. Templates up to 25 nucleotides long were chemically transcribed with an average stepwise yield of 96–97%. The nonenzymatic template-directed generation of primer extension products long enough to encode active ribozymes and/or aptamers inside model protocells suggests possible routes to the synthesis of evolving cellular systems.

Highly efficient editing of theβ-globin gene in patient-derived hematopoietic stem and progenitor cells to treat sickle cell disease.

Sickle cell disease (SCD) is a monogenic disorder that affects millions worldwide. Allogeneic hematopoietic stem cell transplantation is the only available cure. Here, we demonstrate the use of CRISPR/Cas9 and a short single-stranded oligonucleotide template to correct the sickle mutation in the β-globin gene in hematopoietic stem and progenitor cells (HSPCs) from peripheral blood or bone marrow of patients with SCD, with 24.5 ± 7.6% efficiency without selection. Erythrocytes derived from gene-edited cells showed a marked reduction of sickle cells, with the level of normal hemoglobin (HbA) increased to 25.3 ± 13.9%. Gene-corrected SCD HSPCs retained the ability to engraft when transplanted into non-obese diabetic (NOD)-SCID-gamma (NSG) mice with detectable levels of gene correction 16–19 weeks post-transplantation. We show that, by using a high-fidelity SpyCas9 that maintained the same level of on-target gene modification, the off-target effects including chromosomal rearrangements were significantly reduced. Taken together, our results demonstrate efficient gene correction of the sickle mutation in both peripheral blood and bone marrow-derived SCD HSPCs, a significant reduction in sickling of red blood cells, engraftment of gene-edited SCD HSPCs in vivo and the importance of reducing off-target effects; all are essential for moving genome editing based SCD treatment into clinical practice.