On-target DNA Research Articles

T4 DNA polymerase prevents deleterious on-target DNA damage and enhances precise CRISPR editing

Unintended on-target chromosomal alterations induced by CRISPR/Cas9 in mammalian cells are common, particularly large deletions and chromosomal translocations, and present a safety challenge for genome editing. Thus, there is still an unmet need to develop safer and more efficient editing tools. We screened diverse DNA polymerases of distinct origins and identified a T4 DNA polymerase derived from phage T4 that strongly prevents undesired on-target damage while increasing the proportion of precise 1- to 2-base-pair insertions generated during CRISPR/Cas9 editing (termed CasPlus). CasPlus induced substantially fewer on-target large deletions while increasing the efficiency of correcting common frameshift mutations in DMD and restored higher level of dystrophin expression than Cas9-alone in human cardiomyocytes. Moreover, CasPlus greatly reduced the frequency of on-target large deletions during mouse germline editing. In multiplexed guide RNAs mediating gene editing, CasPlus repressed chromosomal translocations while maintaining gene disruption efficiency that was higher or comparable to Cas9 in primary human T cells. Therefore, CasPlus offers a safer and more efficient gene editing strategy to treat pathogenic variants or to introduce genetic modifications in human applications.

Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope

BackgroundNme2ABE8e has been constructed and characterized as a compact, accurate adenine base editor with a less restrictive dinucleotide protospacer-adjacent motif (PAM: N4CC) but low editing efficiency at challenging loci in human cells. Here, we engineered a subset of domain-inlaid Nme2Cas9 base editors to bring the deaminase domain closer to the nontarget strand to improve editing efficiency.ResultsOur results demonstrated that Nme2ABE8e-797 with adenine deaminase inserted between amino acids 797 and 798 has a significantly increased editing efficiency with a wide editing window ranging from 4 to 18 bases in mammalian cells, especially at the sites that were difficult to edit by Nme2ABE8e. In addition, by swapping the PAM-interacting domain of Nme2ABE8e-797 with that of SmuCas9 or introducing point mutations of eNme2-C in Nme2ABE8e-797, we created Nme2ABE8e-797Smu and Nme2ABE8e-797−C, respectively, which exhibited robust activities at a wide range of sites with N4CN PAMs in human cells. Moreover, the modified domain-inlaid Nme2ABE8e can efficiently restore or install disease-related loci in Neuro-2a cells and mice.ConclusionsThese novel Nme2ABE8es with increased on-target DNA editing and expanded PAM compatibility will expand the base editing toolset for efficient gene modification and therapeutic applications.

Engineered lentivirus-derived nanoparticles (LVNPs) for delivery of CRISPR/Cas ribonucleoprotein complexes supporting base editing, prime editing and in vivo gene modification.

Implementation of therapeutic in vivo gene editing using CRISPR/Cas relies on potent delivery of gene editing tools. Administration of ribonucleoprotein (RNP) complexes consisting of Cas protein and single guide RNA (sgRNA) offers short-lived editing activity and safety advantages over conventional viral and non-viral gene and RNA delivery approaches. By engineering lentivirus-derived nanoparticles (LVNPs) to facilitate RNP delivery, we demonstrate effective administration of SpCas9 as well as SpCas9-derived base and prime editors (BE/PE) leading to gene editing in recipient cells. Unique Gag/GagPol protein fusion strategies facilitate RNP packaging in LVNPs, and refinement of LVNP stoichiometry supports optimized LVNP yield and incorporation of therapeutic payload. We demonstrate near instantaneous target DNA cleavage and complete RNP turnover within 4 days. As a result, LVNPs provide high on-target DNA cleavage and lower levels of off-target cleavage activity compared to standard RNP nucleofection in cultured cells. LVNPs accommodate BE/sgRNA and PE/epegRNA RNPs leading to base editing with reduced bystander editing and prime editing without detectable indel formation. Notably, in the mouse eye, we provide the first proof-of-concept for LVNP-directed in vivo gene disruption. Our findings establish LVNPs as promising vehicles for delivery of RNPs facilitating donor-free base and prime editing without formation of double-stranded DNA breaks.

Application of CRISPR-Cas9 System to Study Biological Barriers to Drug Delivery.

In recent years, sequence-specific clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems have been widely used in genome editing of various cell types and organisms. The most developed and broadly used CRISPR-Cas system, CRISPR-Cas9, has benefited from the proof-of-principle studies for a better understanding of the function of genes associated with drug absorption and disposition. Genome-scale CRISPR-Cas9 knockout (KO) screen study also facilitates the identification of novel genes in which loss alters drug permeability across biological membranes and thus modulates the efficacy and safety of drugs. Compared with conventional heterogeneous expression models or other genome editing technologies, CRISPR-Cas9 gene manipulation techniques possess significant advantages, including ease of design, cost-effectiveness, greater on-target DNA cleavage activity and multiplexing capabilities, which makes it possible to study the interactions between membrane proteins and drugs more accurately and efficiently. However, many mechanistic questions and challenges regarding CRISPR-Cas9 gene editing are yet to be addressed, ranging from off-target effects to large-scale genetic alterations. In this review, an overview of the mechanisms of CRISPR-Cas9 in mammalian genome editing will be introduced, as well as the application of CRISPR-Cas9 in studying the barriers to drug delivery.

Targeting DNA polymerase to DNA double-strand breaks reduces DNA deletion size and increases templated insertions generated by CRISPR/Cas9.

Most insertions or deletions generated by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) endonucleases are short (<25 bp), but unpredictable on-target long DNA deletions (>500 bp) can be observed. The possibility of generating long on-target DNA deletions poses safety risks to somatic genome editing and makes the outcomes of genome editing less predictable. Methods for generating refined mutations are desirable but currently unavailable. Here, we show that fusing Escherichia coli DNA polymerase I or the Klenow fragment to Cas9 greatly increases the frequencies of 1-bp deletions and decreases >1-bp deletions or insertions. Importantly, doing so also greatly decreases the generation of long deletions, including those >2 kb. In addition, templated insertions (the insertion of the nucleotide 4 nt upstream of the protospacer adjacent motif) were increased relative to other insertions. Counteracting DNA resection was one of the mechanisms perturbing deletion sizes. Targeting DNA polymerase to double-strand breaks did not increase off-targets or base substitution rates around the cleavage sites, yet increased editing efficiency in primary cells. Our strategy makes it possible to generate refined DNA mutations for improved safety without sacrificing efficiency of genome editing.

Structural basis for mismatch surveillance by CRISPR\u2013Cas9

CRISPR–Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage1–4, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood5–7. Although Cas9 variants with greater discrimination against mismatches have been designed8–10, these suffer from substantially reduced rates of on-target DNA cleavage5,11. Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA–DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA–DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants.

CRISPR/Cas9-based multiplex genome editing of BCL11A and HBG efficiently induces fetal hemoglobin expression

Beta-hemoglobinopathies are caused by mutations in the β-globin gene. One strategy to cure this disease relies on re-activating the γ-globin expression. BCL11A is an important transcription factor that suppresses the γ-globin expression, which makes it one of the most promising therapeutic targets in β-hemoglobinopathies. Here, we performed single-gene editing and multiplex gene editing via CRISPR/Cas9 technology to edit BCL11A erythroid-specific enhancer and BCL11A binding site on γ-globin gene promoter in HUDEP-2 cells and adult human CD34+ cells. Multiplex gene editing led to higher γ-globin expression than single-gene editing without inhibiting erythroid differentiation. By further optimizing the on-target DNA editing efficiency of multiplex gene editing, the percentage of F-cells exceeded 50% in HUDEP-2 cells.Amplicon deep sequencing and whole genome sequencing were used to detect the editing frequency of on- and potential off-target sites in CD34+ cells. No off-target mutations were detected, suggesting its accuracy in HSPCs. In summary, our study provides a new approach which can be used for the treatment of β-hemoglobinopathies in the future.

3027 – GENERATION OF A CD43 FLUORESCENT REPORTER IPSC LINE TO TRACK HEMATOPOIETIC DEVELOPMENT

Our group recently reported enhanced erythroid differentiation from human iPSC by closer mimicry of physiological cell contacts in a three dimensional network, termed „haematopoietic cell forming complex“ (HCFC)1. From this HCFC, CD43+ hematopoietic cells (purity >95 %) were continuously released into the culture supernatant. Characterization of the HCFC in more detail is currently limited by accompanying destruction of the complex, hindering further haematopoietic cell fate tracking. To overcome this limitation, we developed a CD43 fluorescence reporter hiPSC line that allows live-cell imaging of haematopoietic cells as they emerge in a spatiotemporal manner from the HCFC. We have successfully performed gene knock-in of a CD43 promoter region tagged with GFP using CRISPR/Cas9-mediated homology-directed repair (HDR) mechanisms in the AAVS1 safe harbor locus. Adeno-associated viral vectors (AAV) were used for efficient DNA donor delivery into hiPSCs. Following HDR-mediated donor integration, clones with on-target seamless DNA integration were isolated, which was confirmed by Sanger sequencing and in-out PCR. To confirm the functionality of the CD43 fluorescent reporter, haematopoietic and erythroid differentiation of the CD43R-iPSC was induced1. GFP-expression of CD43+ hematopoietic cells inside the HCFC was monitored by fluorescence microscopy and live cell imaging. Haematopoietic nature of released GFP+ cells was confirmed by flow cytometry (CD43, CD34, CD45) and colony formation assay. The established CD43 fluorescent reporter system allows to track hematopoietic development from hiPSCs within three-dimensional structures like organoids. Therefore, the system represents an attractive tool to investigate the hematopoietic development from hiPSCs.The established system can be used for different hiPSC lines or other primary cells. Our group recently reported enhanced erythroid differentiation from human iPSC by closer mimicry of physiological cell contacts in a three dimensional network, termed „haematopoietic cell forming complex“ (HCFC)1. From this HCFC, CD43+ hematopoietic cells (purity >95 %) were continuously released into the culture supernatant. Characterization of the HCFC in more detail is currently limited by accompanying destruction of the complex, hindering further haematopoietic cell fate tracking. To overcome this limitation, we developed a CD43 fluorescence reporter hiPSC line that allows live-cell imaging of haematopoietic cells as they emerge in a spatiotemporal manner from the HCFC. We have successfully performed gene knock-in of a CD43 promoter region tagged with GFP using CRISPR/Cas9-mediated homology-directed repair (HDR) mechanisms in the AAVS1 safe harbor locus. Adeno-associated viral vectors (AAV) were used for efficient DNA donor delivery into hiPSCs. Following HDR-mediated donor integration, clones with on-target seamless DNA integration were isolated, which was confirmed by Sanger sequencing and in-out PCR. To confirm the functionality of the CD43 fluorescent reporter, haematopoietic and erythroid differentiation of the CD43R-iPSC was induced1. GFP-expression of CD43+ hematopoietic cells inside the HCFC was monitored by fluorescence microscopy and live cell imaging. Haematopoietic nature of released GFP+ cells was confirmed by flow cytometry (CD43, CD34, CD45) and colony formation assay. The established CD43 fluorescent reporter system allows to track hematopoietic development from hiPSCs within three-dimensional structures like organoids. Therefore, the system represents an attractive tool to investigate the hematopoietic development from hiPSCs.The established system can be used for different hiPSC lines or other primary cells.

An assessment of genome-editing efficiency of a newly developed Cas9 in C. elegans.

Minimizing the off-target effects of the programmable genome editing tool CRISPR/Cas9 is critical for its potential therapeutic applications. A recent study reported a new Cas9 variant, SuperFi-Cas9, that dramatically reduces off-target DNA cleavage while retaining on-target DNA cleavage activity in in vitro assays. Here we evaluated the genome editing potential of SuperFi-Cas9 by examining its efficiency in mutagenizing targeted genes in the nematode C. elegans . We found that gene mutagenesis rates induced by SuperFi-Cas9 through either non-homologous end joining or homology-directed repair were much lower than those by the wild type Cas9, indicating that Super-Cas9 had very low in vivo DNA cleavage activity. Our results also suggest that C. elegans may serve as an excellent model system for assessing in vivo genome-editing efficiency of newly-developed Cas9 variants.

Comparative structural and dynamics study of free and gRNA-bound FnCas9 and SpCas9 proteins

The introduction of CRISPR/Cas9 based gene editing has greatly accelerated therapeutic genome editing. However, the off-target DNA cleavage by CRISPR/Cas9 protein hampers its clinical translation, hindering its widespread use as a programmable genome editing tool. Although Cas9 variants with better mismatch discrimination have been developed, they have significantly lower rates of on-target DNA cleavage. Here, we have compared the dynamics of a more specific naturally occurring Cas9 from Francisella novicida (FnCas9) to the most widely used, SpCas9 protein. Long-scale atomistic MD simulation of free and gRNA bound forms of both the Cas9 proteins was performed, and their domain rearrangements and binding affinity with gRNA were compared to decipher the possible reason behind the enhanced specificity of FnCas9 protein. The greater binding affinity with gRNA, high domain electrostatics, and more volatility of FnCas9 than SpCas9 may explain its increased specificity and lower tolerance for mismatches.

Updated Results of a Phase 1/2 Clinical Study of Zinc Finger Nuclease-Mediated Editing of BCL11A in Autologous Hematopoietic Stem Cells for Transfusion-Dependent Beta Thalassemia

Updated Results of a Phase 1/2 Clinical Study of Zinc Finger Nuclease-Mediated Editing of BCL11A in Autologous Hematopoietic Stem Cells for Transfusion-Dependent Beta Thalassemia

CABE-RY: A PAM-flexible dual-mutation base editor for reliable modeling of multi-nucleotide variants

Multi-nucleotide variants (MNVs) represent an important type of genetic variation and have biological and clinical significance. To simulate MNVs, we designed four dual-mutation base editors combining hA3A(Y130F), TadA8e(V106W), and protospacer adjacent motif (PAM)-flexible SpRY and selected cytosine and adenine base editor-SpRY (CABE-RY), which had the best editing performance, for further study. Characterization and comparison showed that CABE-RY had a smaller DNA editing window and lower RNA off-target edits than the corresponding single base editors. Thus, we have established a versatile tool to efficiently simulate MNVs over the genome, which could be very useful for functional studies on MNVs in humans.

New targets acquired: Improving locus recovery from the Angiosperms353 probe set.

PREMISEUniversal target enrichment kits maximize utility across wide evolutionary breadth while minimizing the number of baits required to create a cost‐efficient kit. The Angiosperms353 kit has been successfully used to capture loci throughout the angiosperms, but the default target reference file includes sequence information from only 6–18 taxa per locus. Consequently, reads sequenced from on‐target DNA molecules may fail to map to references, resulting in fewer on‐target reads for assembly, and reducing locus recovery.METHODSWe expanded the Angiosperms353 target file, incorporating sequences from 566 transcriptomes to produce a ‘mega353’ target file, with each locus represented by 17–373 taxa. This mega353 file is a drop‐in replacement for the original Angiosperms353 file in HybPiper analyses. We provide tools to subsample the file based on user‐selected taxon groups, and to incorporate other transcriptome or protein‐coding gene data sets.RESULTSCompared to the default Angiosperms353 file, the mega353 file increased the percentage of on‐target reads by an average of 32%, increased locus recovery at 75% length by 49%, and increased the total length of the concatenated loci by 29%.DISCUSSIONIncreasing the phylogenetic density of the target reference file results in improved recovery of target capture loci. The mega353 file and associated scripts are available at: https://github.com/chrisjackson‐pellicle/NewTargets.

CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing.

A substantial fraction of the human genome is difficult to interrogate with short-read DNA sequencing technologies due to paralogy, complex haplotype structures, or tandem repeats. Long-read sequencing technologies, such as Oxford Nanopore's MinION, enable direct measurement of complex loci without introducing many of the biases inherent to short-read methods, though they suffer from relatively lower throughput. This limitation has motivated recent efforts to develop amplification-free strategies to target and enrich loci of interest for subsequent sequencing with long reads. Here, we present CaBagE, a method for target enrichment that is efficient and useful for sequencing large, structurally complex targets. The CaBagE method leverages the stable binding of Cas9 to its DNA target to protect desired fragments from digestion with exonuclease. Enriched DNA fragments are then sequenced with Oxford Nanopore's MinION long-read sequencing technology. Enrichment with CaBagE resulted in a median of 116X coverage (range 39-416) of target loci when tested on five genomic targets ranging from 4-20kb in length using healthy donor DNA. Four cancer gene targets were enriched in a single reaction and multiplexed on a single MinION flow cell. We further demonstrate the utility of CaBagE in two ALS patients with C9orf72 short tandem repeat expansions to produce genotype estimates commensurate with genotypes derived from repeat-primed PCR for each individual. With CaBagE there is a physical enrichment of on-target DNA in a given sample prior to sequencing. This feature allows adaptability across sequencing platforms and potential use as an enrichment strategy for applications beyond sequencing. CaBagE is a rapid enrichment method that can illuminate regions of the 'hidden genome' underlying human disease.

Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene

Delivery of the CRISPR/Cas9 components to the plant cells is a key step in its application as a genome editing tool. Here, we compared Agrobacterium-mediated transformation and protoplast transfection with CRISPR/Cas9 components for potato genome editing. Two sgRNAs were designed to simultaneously direct Cas9 to the StPPO2 gene encoding for a tuber polyphenol oxidase (PPO). A binary vector (CR-PPO) was utilized for either Agrobacterium-mediated transformation or for transient expression in protoplasts, while ribonucleoprotein complexes (RNP-PPO) were additionally assayed in protoplasts. Editing efficiency varied, yielding 9.6%, 18.4% and 31.9% of edited lines from Agrobacterium-mediated transformation, RNP-PPO and CR-PPO transient expression in protoplasts, respectively. Furthermore, only the CR-PPO transient expression resulted in lines edited in all four StPPO2 alleles, observed in 46% of the edited lines and confirmed by tuber PPO activity and enzymatic browning analysis. Lines with on-target DNA insertions were found from all three approaches and characterized by sequencing. The dual-sgRNA strategy resulted in a low incidence of the targeted deletion, likely due to contrasting efficiencies between sgRNAs, that was partially evident in the in silico analysis. Our results demonstrate that gene editing efficiency in potato depends on the CRISPR/Cas9 delivery strategy and provide insights to consider when selecting the appropriate methodology. We compared the StPPO2 gene editing outcomes and efficiencies through Agrobacterium-mediated transformation and protoplasts transfection with DNA or RNPs, and demonstrated that genome editing efficiency depends on the CRISPR/Cas9 delivery approach in potato.

Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing

Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.

Preliminary Results of a Phase 1/2 Clinical Study of Zinc Finger Nuclease-Mediated Editing of BCL11A in Autologous Hematopoietic Stem Cells for Transfusion-Dependent Beta Thalassemia

Introduction: Persistently high fetal hemoglobin (HbF) expression can ameliorate severe transfusion-dependent beta thalassemia (TDT). BCL11A, a master regulator of the fetal-to-adult hemoglobin switch, is a rational gene-editing target in beta globinopathies. In pre-clinical studies with human hematopoietic stem cells (HSC), zinc finger nuclease (ZFN)-mediated disruption of the GATA-binding region of the intronic erythroid-specific enhancer (BCL11A ESE) increased endogenous HbF production in erythroid cells while allowing healthy, multi-lineage hematopoiesis. Though allogeneic hematopoietic stem cell transplantation (HSCT) can be curative in TDT, its application is partly limited by donor availability. Autologous transplantation using ex vivo gene-modified HSCs (HSCGT) can circumvent this, and lentiviral vector-mediated beta globin gene addition studies have shown efficacy in TDT. However, the long-term safety of random lentiviral genomic integration in HSCs is uncertain. ST-400 is an investigational cell therapy comprised of autologous CD34+ cells that have undergone high-precision, ZFN-mediated ex vivo editing at BCL11A ESE. The aim of this study is to induce HbF expression in edited erythroid cells. We hypothesized that HSCGT with ST-400 is safe and effective in TDT. Methods: The Thales trial (NCT03432364) is a Phase I/II study of the safety, tolerability and efficacy of ST-400 in adult patients with TDT, defined as undergoing ≥8 annual packed red blood cell transfusion episodes for at least 2 consecutive years before enrollment. After routine leukapheresis following mobilization with G-CSF and plerixafor, autologous collections are enriched for CD34+ cells and transfected with mRNA encoding ZFNs with binding sites flanking the GATA-binding region of BCL11A ESE. ST-400 product is infused following myeloablative busulfan conditioning. The trial will enroll 6 patients who are monitored for safety and efficacy for 3 years post-infusion. Results: Three patients have completed ST-400 manufacturing, and two have been infused. Patient 1 (β0/β0 genotype) received an ST-400 dose of 6.1 x 106 cells/kg. The patient experienced a serious adverse event (SAE) of hypersensitivity during ST-400 infusion considered to be related to the product cryoprotectant, DMSO, that resolved by the end of infusion. The patient had prompt hematopoietic reconstitution (ANC recovery day +14; platelet recovery day +24), with increasing HbF fraction that contributed to stable total hemoglobin. After being free from PRBC transfusions for 6 weeks, the patient has since required intermittent PRBC transfusions. At last follow-up, on-target DNA insertions-deletions (indels) at BCL11A ESE were present in peripheral blood mononuclear cells (PBMCs), and HbF levels remain elevated at 6 months post-infusion. Patient 2 (homozygous for the severe β+ IVS-I-5 G&amp;gt;C mutation) received an ST-400 dose of 4.5 x 106 cells/kg. There was prompt hematopoietic reconstitution (ANC recovery day +15; platelet recovery day +29) with on-target indels detected in PBMCs at last follow-up, and rising HbF levels observed through 90 days post-infusion. Longer follow-up will be required to assess the clinical significance of these early results. Patient 3 (β0/β+ genotype including the severe IVS-II-654 C&amp;gt;T mutation) has completed ST-400 manufacturing. Besides the SAE reported for Patient 1, no other SAEs related to ST-400 have been reported and other AEs have been consistent with myeloablation. No clonal hematopoiesis has been observed. Conclusions: ST-400 is an ex vivo, ZFN-edited autologous HSC product for increased erythroid HbF expression in TDT. Two infused patients had rapid hematopoietic reconstitution following myeloablative conditioning, and both have elevated HbF levels following HSCGT. These data are preliminary, and additional patients and longer follow-up will be required to understand the safety and efficacy of this therapy. Disclosures Smith: Amgen: Research Funding; Jazz Pharmaceuticals: Research Funding. Schiller:Agios: Research Funding, Speakers Bureau; Eli Lilly and Company: Research Funding; FujiFilm: Research Funding; Genzyme: Research Funding; Gilead: Research Funding; Incyte: Research Funding; J&amp;J: Research Funding; Jazz Pharmaceuticals: Honoraria, Research Funding; Karyopharm: Research Funding; Novartis: Research Funding; Onconova: Research Funding; Pfizer Pharmaceuticals: Equity Ownership, Research Funding; Sangamo Therapeutics: Research Funding; Amgen: Other, Research Funding; Daiichi Sankyo: Research Funding; Constellation Pharmaceutical: Research Funding; Celgene: Research Funding, Speakers Bureau; Bristol Myer Squibb: Research Funding; Biomed Valley Discoveries: Research Funding; Astellas: Research Funding. Vercellotti:Mitobridge, an Astellas Company: Consultancy, Research Funding. Kwiatkowski:bluebird bio, Inc.: Consultancy, Research Funding; Agios: Consultancy; Terumo: Research Funding; Novartis: Research Funding; Apopharma: Research Funding; Imara: Consultancy; Celgene: Consultancy. Williams:bluebird bio: Patents &amp; Royalties: License of certain IP relevant to hemoglobinopathies to bluebird bio. Potential for future royalty/milestone income., Research Funding; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder, Patents &amp; Royalties: Potential for future royalty/milestone income, X-SCID., Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Alerion Biosciences: Other: Co-founder. Miller:Sangamo Therapeutics: Employment, Equity Ownership. Woolfson:Sangamo Therapeutics: Employment, Equity Ownership. Walters:Editas Medicine: Consultancy; TruCode: Consultancy; AllCells, Inc: Consultancy. OffLabel Disclosure: busulfan: used for myeloablation prior to infusing the investigational autologous HSPC product (ST-400) plerixafor: used with G-CSF to enhance mobilization of autologous HSPC for collection via leukapheresis. Autologous HSPC then undergo ex vivo manufacturing to generate the investigational product (ST-400)

Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors.

CRISPR-Cas base-editor technology enables targeted nucleotide alterations, and is being increasingly used for research and potential therapeutic applications1,2. The most widely used cytosine base editors (CBEs) induce deamination of DNA cytosines using the rat APOBEC1 enzyme, which is targeted by a linked Cas protein-guide RNA complex3,4. Previous studies of the specificity of CBEs have identified off-target DNA edits in mammalian cells5,6. Here we show that a CBE with rat APOBEC1 can cause extensive transcriptome-wide deamination of RNA cytosines in human cells, inducing tens of thousands of C-to-U edits with frequencies ranging from 0.07% to 100% in 38-58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, and 5' and 3' untranslated region mutations. We engineered two CBE variants bearing mutations in rat APOBEC1 that substantially decreased the number of RNA edits (by more than 390-fold and more than 3,800-fold) in human cells. These variants also showed more precise on-target DNA editing than the wild-type CBE and, for most guide RNAs tested, no substantial reduction in editing efficiency. Finally, we show that an adenine base editor7 can also induce transcriptome-wide RNA edits. These results have implications for the use of base editors in both research and clinical settings, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.

DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA.

To investigate whether and how CRISPR-Cas9 on-target and off-target activities are affected by chromatin in eukaryotic cells, we first identified a series of identical endogenous DNA sequences present in both open and closed chromatin regions and then measured mutation frequencies at these sites in human cells using Cas9 complexed with matched or mismatched sgRNAs. Unlike matched sgRNAs, mismatched sgRNAs were highly sensitive to chromatin states, suggesting that off-target but not on-target DNA cleavage is hindered by chromatin. We next performed Digenome-seq using cell-free chromatin DNA (now termed DIG-seq) and histone-free genomic DNA in parallel and found that only a subset of sites, cleaved in histone-free DNA, were cut in chromatin DNA, suggesting that chromatin can inhibit Cas9 off-target effects in favor of its genome-wide specificity in cells.

Enhanced Proofreading Governs CRISPR-Cas9 Targeting Accuracy

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule Förster resonance energy transfer (smFRET), we identified an intermediate state of Streptococcus pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking into its active state. We showed that the high-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) Cas9 variants 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 designed 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.