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Genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) is a sensitive, unbiased, genome-wide method for defining the activity of genome-editing nucleases in living cells. GUIDE-seq is based on the principle of efficient integration of an end-protected double-stranded oligodeoxynucleotide tag into sites of nuclease-induced DNA double-stranded breaks, followed by amplification of tag-containing genomic DNA molecules and high-throughput sequencing. Here we describe a detailed GUIDE-seq protocol including cell transfection, library preparation, sequencing and bioinformatic analysis. The entire protocol including cell culture can be completed in 9 d. Once tag-integrated genomic DNA is isolated, library preparation, sequencing and analysis can be performed in 3 d. The result is a genome-wide catalog of off-target sites ranked by nuclease activity as measured by GUIDE-seq read counts. GUIDE-seq is one of the most sensitive cell-based methods for defining genome-wide off-target activity and has been broadly adopted for research and therapeutic use.

Epigenetic editing is an emerging technology that uses artificial transcription factors (aTFs) to regulate expression of a target gene. Although human genes can be robustly upregulated by targeting aTFs to promoters, the activation induced by directing aTFs to distal transcriptional enhancers is substantially less robust and consistent. Here we show that long-range activation using CRISPR-based aTFs in human cells can be made more efficient and reliable by concurrently targeting an aTF to the target gene promoter. We used this strategy to direct target gene choice for enhancers capable of regulating more than one promoter and to achieve allele-selective activation of human genes by targeting aTFs to single-nucleotide polymorphisms embedded in distally located sequences. Our results broaden the potential applications of the epigenetic editing toolbox for research and therapeutics.

Base editing by nucleotide deaminases linked to programmable DNA-binding proteins represents a promising approach to permanently remedy blood disorders, although its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. In this study, we purified A3A (N57Q)-BE3 base editor for ribonucleoprotein (RNP) electroporation of human-peripheral-blood-mobilized CD34+hematopoietic stem and progenitor cells (HSPCs). We observed frequent on-target cytosine base edits at the BCL11Aerythroid enhancer at +58 with few indels. Fetal hemoglobin (HbF) induction in erythroid progeny after base editing or nuclease editing was similar. A single therapeutic base edit of the BCL11Aenhancer prevented sickling and ameliorated globin chain imbalance in erythroid progeny from sickle cell disease and β-thalassemia patient-derived HSPCs, respectively. Moreover, efficient multiplex editing could be achieved with combined disruption of the BCL11Aerythroid enhancer and correction of the HBB−28A>G promoter mutation. Finally, base edits could be produced in multilineage-repopulating self-renewing human HSCs with high frequency as assayed in primary and secondary recipient animals resulting in potent HbF induction in vivo. Together, these results demonstrate the potential of RNP base editing of human HSPCs as a feasible alternative to nuclease editing for HSC-targeted therapeutic genome modification.

Since most dominant human mutations are single nucleotide substitutions1,2, we explored gene editing strategies to disrupt dominant mutations efficiently and selectively without affecting wild-type alleles. However, single nucleotide discrimination can be difficult to achieve3because commonly used endonucleases, such as Streptococcus pyogenesCas9 (SpCas9), can tolerate up to seven mismatches between guide RNA (gRNA) and target DNA. Furthermore, the protospacer-adjacent motif (PAM) in some Cas9 enzymes can tolerate mismatches with the target DNA3,4. To circumvent these limitations, we screened 14 Cas9/gRNA combinations for specific and efficient disruption of a nucleotide substitution that causes the dominant progressive hearing loss, DFNA36. As a model for DFNA36, we used Beethovenmice5, which harbor a point mutation in Tmc1, a gene required for hearing that encodes a pore-forming subunit of mechanosensory transduction channels in inner-ear hair cells6. We identified a PAM variant of Staphylococcus aureusCas9 (SaCas9-KKH) that selectively and efficiently disrupted the mutant allele, but not the wild-type Tmc1/TMC1allele, in Beethovenmice and in a DFNA36 human cell line. Adeno-associated virus (AAV)-mediated SaCas9-KKH delivery prevented deafness in Beethovenmice up to one year post injection. Analysis of current ClinVar entries revealed that ~21% of dominant human mutations could be targeted using a similar approach.

Eric Lander, Françoise Baylis, Feng Zhang, Emmanuelle Charpentier, Paul Berg and specialists from seven countries call for an international governance framework.

Circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) is a sensitive and unbiased method for defining the genome-wide activity (on-target and off-target) of CRISPR–Cas9 nucleases by selective sequencing of nuclease-cleaved genomic DNA (gDNA). Here, we describe a detailed experimental and analytical protocol for CIRCLE-seq. The principle of our method is to generate a library of circularized gDNA with minimized numbers of free ends. Highly purified gDNA circles are treated with CRISPR–Cas9 ribonucleoprotein complexes, and nuclease-linearized DNA fragments are then ligated to adapters for high-throughput sequencing. The primary advantages of CIRCLE-seq as compared with other in vitro methods for defining genome-wide genome editing activity are (i) high enrichment for sequencing nuclease-cleaved gDNA/low background, enabling sensitive detection with low sequencing depth requirements; and (ii) the fact that paired-end reads can contain complete information on individual nuclease cleavage sites, enabling use of CIRCLE-seq in species without high-quality reference genomes. The entire protocol can be completed in 2 weeks, including time for gRNA cloning, sequence verification, in vitro transcription, library preparation, and sequencing.

CRISPR–Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications1–6but identifying unwanted off-target mutations is important for clinical translation7. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe ‘verification of in vivo off-targets’ (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR–Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR–Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.

Supplemental Digital Content is available in the text.

Inducible dCpf1-based transcriptional activators achieve tunable, multiplexed target gene expression.

The RNA-guided CRISPR–Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity are unknown. Here, using single-molecule Förster resonance energy transfer experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we design a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

Deficiency of factor X (F10) in humans is a rare bleeding disorder with a heterogeneous phenotype and limited therapeutic options. Targeted disruption of F10 and other common pathway factors in mice results in embryonic/neonatal lethality with rapid resorption of homozygous mutants, hampering additional studies. Several of these mutants also display yolk sac vascular defects, suggesting a role for thrombin signaling in vessel development. The zebrafish is a vertebrate model that demonstrates conservation of the mammalian hemostatic and vascular systems. We have leveraged these advantages for in-depth study of the role of the coagulation cascade in the developmental regulation of hemostasis and vasculogenesis. In this article, we show that ablation of zebrafish f10 by using genome editing with transcription activator-like effector nucleases results in a major embryonic hemostatic defect. However, widespread hemorrhage and subsequent lethality does not occur until later stages, with absence of any detectable defect in vascular development. We also use f10−/− zebrafish to confirm 5 novel human F10 variants as causative mutations in affected patients, providing a rapid and reliable in vivo model for testing the severity of F10 variants. These findings as well as the prolonged survival of f10−/− mutants will enable us to expand our understanding of the molecular mechanisms of hemostasis, including a platform for screening variants of uncertain significance in patients with F10 deficiency and other coagulation disorders. Further study as to how fish tolerate what is an early lethal mutation in mammals could facilitate improvement of diagnostics and therapeutics for affected patients with bleeding disorders.

Sensitive detection of off-target effects is important for translating CRISPR–Cas9 nucleases into human therapeutics. In vitro biochemical methods for finding off-targets offer the potential advantages of greater reproducibility and scalability while avoiding limitations associated with strategies that require the culture and manipulation of living cells. Here we describe circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochemical approaches for identifying CRISPR–Cas9 genome-wide off-target mutations. In contrast to previously described in vitro methods, we show that CIRCLE-seq can be practiced using widely accessible next-generation sequencing technology and does not require reference genome sequences. Importantly, CIRCLE-seq can be used to identify off-target mutations associated with cell-type-specific single-nucleotide polymorphisms, demonstrating the feasibility and importance of generating personalized specificity profiles. CIRCLE-seq provides an accessible, rapid, and comprehensive method for identifying genome-wide off-target mutations of CRISPR–Cas9.

CRISPR–Cas9 RNA-guided nucleases are a transformative technology for biology, genetics and medicine owing to the simplicity with which they can be programmed to cleave specific DNA target sites in living cells and organisms. However, to translate these powerful molecular tools into safe, effective clinical applications, it is of crucial importance to carefully define and improve their genome-wide specificities. Here, we outline our state-of-the-art understanding of target DNA recognition and cleavage by CRISPR–Cas9 nucleases, methods to determine and improve their specificities, and key considerations for how to evaluate and reduce off-target effects for research and therapeutic applications.

CRISPR–Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenesCas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.

Pathologic blood clotting is a leading cause of morbidity and mortality in the developed world, underlying deep vein thrombosis, myocardial infarction, and stroke. Genetic predisposition to thrombosis is still poorly understood, and we hypothesize that there are many additional risk alleles and modifying factors remaining to be discovered. Mammalian models have contributed to our understanding of thrombosis, but are low throughput and costly. We have turned to the zebrafish, a tool for high-throughput genetic analysis. Using zinc finger nucleases, we show that disruption of the zebrafish antithrombin III (at3) locus results in spontaneous venous thrombosis in larvae. Although homozygous mutants survive into early adulthood, they eventually succumb to massive intracardiac thrombosis. Characterization of null fish revealed disseminated intravascular coagulation in larvae secondary to unopposed thrombin activity and fibrinogen consumption, which could be rescued by both human and zebrafish at3 complementary DNAs. Mutation of the human AT3-reactive center loop abolished the ability to rescue, but the heparin-binding site was dispensable. These results demonstrate overall conservation of AT3 function in zebrafish, but reveal developmental variances in the ability to tolerate excessive clot formation. The accessibility of early zebrafish development will provide unique methods for dissection of the underlying mechanisms of thrombosis.

Pathologic blood clotting is a leading cause of morbidity and mortality in the developed world, underlying deep vein thrombosis, myocardial infarction, and stroke. Genetic predisposition to thrombosis is still poorly understood, and we hypothesize that there are many additional risk alleles and modifying factors remaining to be discovered. Mammalian models have contributed to our understanding of thrombosis, but are low throughput and costly. We have turned to the zebrafish, a tool for high-throughput genetic analysis. Using zinc finger nucleases, we show that disruption of the zebrafish antithrombin III (at3) locus results in spontaneous venous thrombosis in larvae. Although homozygous mutants survive into early adulthood, they eventually succumb to massive intracardiac thrombosis. Characterization of null fish revealed disseminated intravascular coagulation in larvae secondary to unopposed thrombin activity and fibrinogen consumption, which could be rescued by both human and zebrafish at3complementary DNAs. Mutation of the human AT3-reactive center loop abolished the ability to rescue, but the heparin-binding site was dispensable. These results demonstrate overall conservation of AT3 function in zebrafish, but reveal developmental variances in the ability to tolerate excessive clot formation. The accessibility of early zebrafish development will provide unique methods for dissection of the underlying mechanisms of thrombosis.

Although many distinct mutations in a variety of genes are known to cause Amyotrophic Lateral Sclerosis (ALS), it remains poorly understood how they selectively impact motor neuron biology and whether they converge on common pathways to cause neuronal degeneration. Here, we have combined reprogramming and stem cell differentiation approaches with genome engineering and RNA sequencing to define the transcriptional and functional changes that are induced in human motor neurons by mutant SOD1. Mutant SOD1 protein induced a transcriptional signature indicative of increased oxidative stress, reduced mitochondrial function, altered subcellular transport, and activation of the ER stress and unfolded protein response pathways. Functional studies demonstrated that these pathways were perturbed in a manner dependent on the SOD1mutation. Finally, interrogation of stem-cell-derived motor neurons produced from ALS patients harboring a repeat expansion in C9orf72indicates that at least a subset of these changes are more broadly conserved in ALS.

Targeting the BCL11Aerythroid enhancer by gene editing is a promising approach to fetal hemoglobin induction for beta-hemoglobinopathies. HbF levels vary widely among individuals, suggesting potential heterogeneity in HbF responses after therapeutic intervention. We hypothesize that maximizing both gene edit frequency and HbF induction potential could promote consistently favorable clinical outcomes. Here we compared CRISPR-Cas9 endonuclease editing of the BCL11A+58 enhancer with alternative gene modification approaches, including +55 erythroid enhancer editing alone or in combination with the +58 enhancer, as well as editing targeting the HBG1/2promoter -115 BCL11A binding site and transduction by an shRNA knocking down the BCL11A transcript in erythroid precursors. We found that combined targeting of the BCL11A+58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in the most potent HbF induction (52.4%±6.3%) of tested approaches (BCL11A+58 editing alone, 29.1%±3.9%; BCL11A+55 editing alone, 34.8±5.1%; HBG1/2promoter editing, 34.1% ±5.4%; shmiR-BCL11A, 32.2%±4.4%; mock, 7.6%±3.4%). Based on assays in bulk and single cell derived erythroid cultures and xenografted immunodeficient mice, we found that disruption of core half E-box/GATA motifs at both the +58 and +55 enhancers was associated with greatest HbF induction, whether by small indels, interstitial 3.1 kb deletion, or 3.1 kb inversion. Rare gene edited clones with alleles that only partially disrupted these motifs were associated with intermediate HbF induction phenotypes. Combined editing of BCL11A+58 and +55 enhancers was compatible with HSC self-renewal in primary and secondary xenotransplant, with intact lymphoid, myeloid and erythroid repopulation. We conducted gene-edited cell product manufacturing process development and developed conditions using a MaxCyte electroporation instrument achieving mean 97.3±1.8% gene edits and 88.9%±6.4% viability 24 hours after electroporation in 3 engineering runs at clinical scale. We obtained similar results at small-scale with plerixafor-mobilized HSPCs from sickle cell disease (SCD) donors or G-CSF mobilized PBMCs from transfusion-dependent beta-thalassemia (TDT) donors, including 94.2%±4.4%, 99.5%±0.3% and 91.8%±6.3% of gene edits in engrafting cells from NBSGW 16 week mouse bone marrow of healthy, SCD and TDT donors respectively. Off-target analyses by pooled amplicon sequencing of 601 candidate off-target sites for the +58 and +55 targeting sgRNAs, nominated by a range of computational (CRISPRme) and experimental (GUIDE-seq and ONE-seq) methods, did not identify reference genome off-target edits at a sensitivity of 0.1% allele frequency. We evaluated +58/+55 enhancer combined targeting in nonhuman primates by performing ribonucleoprotein (RNP) electroporation in rhesus macaque mobilized peripheral blood CD34+ HSPCs with autologous re-infusion following busulfan myeloablation. We observed highly efficient gene edit frequency (85.2%, 88.8% and 84.9%) and durable HbF induction (26.4%, 57.5%, and 45.9% F-cells and 12.7%, 41.9%, and 28% gamma-globin) in the peripheral blood in 3 animals at most recent recorded time point post infusion (127, 78, and 54 weeks respectively). Single colony analyses and bulk ddPCR and unidirectional sequencing demonstrated that the long-term engrafting cells displayed a similar distribution of indels, 3.1 kb deletions, and 3.1 kb inversions as the input cell products. Erythroid stress due to hydroxyurea treatment, with or without phlebotomy, was associated with substantially augmented HbF responses (to 75.9%, 88.2%, and 57.8% F-cells and 47.9%, 68%, and 35.7% gamma-globin). No hematologic or other toxicities attributable to gene editing were observed.

No relevant conflicts of interest to declare.

Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using “modular assembly” have been described, standardized reagents and protocols that permit rapid, cross-platform “mixing-and-matching” of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest. Our reagents have been standardized on a single platform, enabling facile mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design. We also describe a bacterial cell-based reporter assay for rapidly screening the DNA-binding activities of assembled multi-finger arrays. This protocol can be completed in approximately 24–26 d.

Targeted genome editing using engineered nucleases can reliably generate defined genetic mutations, although the range of off-target lesions caused by these techniques is unclear. Recent papers in Cell Stem Cellperform high-coverage whole-genome sequencing to demonstrate the feasibility of obtaining correctly targeted pluripotent stem cell clones with minimal unintended modifications.

Joung: Horizon Discovery: Consultancy; Editas Medicine: Other: Financial interest; Poseida Therapeutics: Other: Financial interest; Hera Testing Laboratories: Other: Financial interest; Transposagen Biopharmaceuticals: Other: Financial interest. Shavit:CSL Behring: Consultancy; Octapharma: Consultancy; Bayer: Consultancy; Baxter: Consultancy.

Factor X (F10) deficiency is a rare inherited bleeding disorder with a heterogeneous phenotype and limited therapeutic options. Targeted knockout of F10and other common pathway factors in mice results in embryonic/neonatal lethality with rapid resorption of homozygous mutants, hampering further studies. Several of these mutants also display yolk sac vascular defects, suggesting a role for thrombin signaling in development. The zebrafish model is characterized by external development, optical transparency, ability to generate thousands of offspring at low cost, and a highly characterized vasculature. We have used these advantages for more in depth study of the role of the coagulation cascade in developmental regulation of hemostasis and vasculogenesis.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated (Cas) system has been broadly adopted for highly efficient genome editing in a variety of model organisms and human cell types. Unlike previous genome editing technologies such as zinc finger nucleases (ZFNs) and transcription activator−like effector nucleases (TALENs), CRISPR/Cas technology does not require complex protein engineering and can be utilized by any researcher proficient in basic molecular biology and cell culture techniques. This unit describes protocols for design and cloning of vectors expressing single or multiplex gRNAs, for transient transfection of human cell lines, and for quantitation of mutation frequencies by T7 endonuclease I assay. These protocols also include guidance for using two improvements that increase the specificity of CRISPR/Cas nucleases: truncated gRNAs and dimeric RNA‐guided FokI nucleases. © 2015 by John Wiley & Sons, Inc.

Joung: Editas Medicine: Consultancy, Equity Ownership, Other, Patents & Royalties; Transposagen Biopharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Horizon Discovery: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Targeted genome and epigenome editing technologies have recently emerged as important tools for biomedical research and as potential reagents for therapies of gene-based diseases. This talk will focus on our recent work on both improving and defining the genome wide specificities of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas nucleases, a robust platform for introducing targeted genome sequence alterations. The talk will also briefly describe the creation and validation of new technologies for modifying specific epigenomic marks on histones and DNA that can be used to induce targeted alterations in endogenous human gene expression. Taken together, these methodologies provide transformative tools for understanding human biology and offer promising pathways forward for developing new classes of therapeutics based on targeted alterations of gene sequence and/or gene expression.

No relevant conflicts of interest to declare.

Thrombosis is a leading cause of morbidity and mortality in the developed world, underlying deep vein thrombosis, myocardial infarction, and stroke. Identification of small molecule inhibitors of thrombosis in an in vivomodel would facilitate novel and improved therapeutics for patients. The zebrafish is a powerful genetic model in which the hemostatic system is nearly entirely conserved with humans. Its external development, ability to generate thousands of offspring at low cost, and optical transparency all make it a powerful tool to screen for genetic and chemical modifiers of coagulation disorders. We generated a zebrafish model of antithrombin III (AT3) deficiency by targeted mutagenesis using zinc finger nucleases. Homozygous at3mutants displayed a lethal phenotype due to intracardiac thrombosis between 2 and 7 months of age, yet embryos and larvae appeared grossly normal with no overt evidence of pathologic clotting. Induction of thrombosis at 3-4 days post fertilization (dpf) in homozygous mutant larvae by laser-mediated endothelial ablation resulted in diminished rates of posterior cardinal vein (PCV) occlusion, a bleeding phenotype. To prove functional conservation with mammals, we expressed recombinant zebrafish At3 and demonstrated that it binds human thrombin in vitro. Furthermore, while injection of wild type zebrafish and human cDNAs rescued the laser injury phenotype, zebrafish at3with a mutation in the putative reactive site failed to do so.

Engineered transcription activator–like effector nucleases (TALENs) are broadly useful tools for performing targeted genome editing in a wide variety of organisms and cell types including plants, zebrafish, C. elegans, rat, human somatic cells, and human pluripotent stem cells. Here we describe detailed protocols for the serial, hierarchical assembly of TALENs that require neither PCR nor specialized multi‐fragment ligations and that can be implemented by any laboratory. These restriction enzyme and ligation (REAL)–based protocols can be practiced using plasmid libraries and user‐friendly, Web‐based software that both identifies target sites in sequences of interest and generates printable graphical guides that facilitate assembly of TALENs. With the described platform of reagents, protocols, and software, researchers can easily engineer multiple TALENs within 2 weeks using standard cloning techniques. Curr. Protoc. Mol. Biol. 100:12.15.1‐12.15.14. © 2012 by John Wiley & Sons, Inc.