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Rhabdomyosarcoma (RMS) is a common childhood cancer that shares features with developing skeletal muscle. Yet, the conservation of cellular hierarchy with human muscle development and the identification of molecularly defined tumor-propagating cells has not been reported. Using single-cell RNA-sequencing, DNA-barcode cell fate mapping and functional stem cell assays, we uncovered shared tumor cell hierarchies in RMS and human muscle development. We also identified common developmental stages at which tumor cells become arrested. Fusion-negative RMS cells resemble early myogenic cells found in embryonic and fetal development, while fusion-positive RMS cells express a highly specific gene program found in muscle cells transiting from embryonic to fetal development at 7-7.75 weeks of age. Fusion-positive RMS cells also have neural pathway-enriched states, suggesting less-rigid adherence to muscle-lineage hierarchies. Finally, we identified a molecularly defined tumor-propagating subpopulation in fusion-negative RMS that shares remarkable similarity to bi-potent, muscle mesenchyme progenitors that can make both muscle and osteogenic cells.

SUMMARYGene editing theBCL11Aerythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here we compared combined CRISPR-Cas9 endonuclease editing of theBCL11A+58 and +55 enhancers with leading gene modification approaches under clinical investigation. We found that combined targeting of theBCL11A+58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in superior HbF induction, including in engrafting erythroid cells from sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. We corroborated prior observations that double strand breaks (DSBs) could produce unintended on- target outcomes in hematopoietic stem and progenitor cells (HSPCs) such as long deletions and centromere-distal chromosome fragment loss. We show these unintended outcomes are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing HSPCs without cytokine culture bypassed long deletion and micronuclei formation while preserving efficient on-target editing and engraftment function. These results indicate that nuclease editing of quiescent hematopoietic stem cells (HSCs) limits DSB genotoxicity while maintaining therapeutic potency and encourages efforts for in vivo delivery of nucleases to HSCs.

MYCderegulation occurs in 67% of multiple myeloma (MM) cases and associates with progression and worse prognosis in MM. EnhancedMYCexpression is known to be driven by translocation or amplification events, but it only occurs in 40% of MM patients. Here, we describe a new mechanism ofMYCregulation, whereby epigenetic regulation ofMYCby increased accessibility of a cell-type specific enhancer leads to increasedMYCexpression. We found enhancer activity does not associate with enhancer hijacking events. We identified specific binding of c-MAF, IRF4, and SPIB transcription factors to the enhancer can activateMYC. In addition, we discovered focal amplification of this specific enhancer in approximately 4% of MM patients. Together, our findings define a new epigenetic mechanism ofMYCderegulation in MM beyond known translocations or amplifications and point to the importance of non-coding regulatory elements and their associated transcription factor networks as drivers of MM progression.

Cas9 is a programmable nuclease that has furnished transformative technologies, including base editors and transcription modulators (e.g., CRISPRi/a), but several applications of these technologies, including therapeutics, mandatorily require precision control of their half-life. For example, such control can help avert any potential immunological and adverse events in clinical trials. Current genome editing technologies to control the half-life of Cas9 are slow, have lower activity, involve fusion of large response elements (> 230 amino acids), utilize expensive controllers with poor pharmacological attributes, and cannot be implementedin vivoon several CRISPR-based technologies. We report a general platform for half-life control using the molecular glue, pomalidomide, that binds to a ubiquitin ligase complex and a response-element bearing CRISPR-based technology, thereby causing the latter’s rapid ubiquitination and degradation. Using pomalidomide, we were able to control the half-life of large CRISPR-based technologies (e.g., base editors, CRISPRi) and small anti-CRISPRs that inhibit such technologies, allowing us to build the first examples of on-switch for base editors. The ability to switch on, fine-tune and switch-off CRISPR-based technologies with pomalidomide allowed complete control over their activity, specificity, and genome editing outcome. Importantly, the miniature size of the response element and favorable pharmacological attributes of the drug pomalidomide allowed control of activity of base editorin vivousing AAV as the delivery vehicle. These studies provide methods and reagents to precisely control the dosage and half-life of CRISPR-based technologies, propelling their therapeutic development.

CRISPR gene editing holds great promise to modify DNA sequences in somatic cells to treat disease. However, standard computational and biochemical methods to predict off-target potential focus on reference genomes. We developed an efficient tool called CRISPRme that considers SNP and indel genetic variants to nominate and prioritize off-target sites. We tested the software with a BCL11A enhancer targeting guide RNA showing promise in clinical trials for sickle cell disease and β-thalassemia and found that the top candidate off-target is produced by an allele common in African-ancestry populations (MAF 4.5%) that introduces a protospacer adjacent motif (PAM) sequence. We validated that SpCas9 generates strictly allele-specific indels and pericentric inversions in CD34+ HSPCs, although high-fidelity Cas9 mitigates this off-target. This report illustrates how genetic variants should be considered as modifiers of gene editing outcomes. We expect that variant-aware off-target assessment will become integral to therapeutic genome editing evaluation and provide a powerful approach for comprehensive off-target nomination.

SummaryAlthough vast numbers of putative gene regulatory elements have been cataloged, the sequence motifs and individual bases that underlie their functions remain largely unknown. Here we combine epigenetic perturbations, base editing, and deep learning models to dissect regulatory sequences within the exemplar immune locus encoding CD69. Focusing on a differentially accessible and acetylated upstream enhancer, we find that the complementary strategies converge on a ∼170 base interval as critical for CD69 induction in stimulated Jurkat T cells. We pinpoint individual cytosine to thymine base edits that markedly reduce element accessibility and acetylation, with corresponding reduction of CD69 expression. The most potent base edits may be explained by their effect on binding competition between the transcriptional activator GATA3 and the repressor BHLHE40. Systematic analysis of GATA and bHLH/Ebox motifs suggests that interplay between these factors plays a general role in rapid T cell transcriptional responses. Our study provides a framework for parsing gene regulatory elements in their endogenous chromatin contexts and identifying operative artificial variants.HighlightsBase editing screens and deep learning pinpoint sequences and single bases affecting immune gene expressionAn artificial C-to-T variant in a regulatory element suppresses CD69 expression by altering the balance of transcription factor bindingCompetition between GATA3 and BHLHE40 regulates inducible immune genes and T cell states

Single-cell RNA Velocity has dramatically advanced our ability to model cellular differentiation and cell fate decisions. However, current preprocessing choices and model assumptions often lead to errors in assigning developmental trajectories. Here, we develop, Pyro-Velocity, a Bayesian, generative, and multivariate RNA Velocity model to estimate the uncertainty of cell future states. This approach models raw sequencing counts with the synchronized cell time across all expressed genes to provide quantifiable and improved information on cell fate choices and developmental trajectory dynamics.

Acute myeloid leukemia (AML) is a devastating disease, and clinical outcomes are still far from satisfactory. Here, to identify novel targets for AML therapy, we performed a genome-wide CRISPR/Cas9 screen using AML cell lines, followed by a second screen in vivo. We show that PAICS, an enzyme involved in de novo purine biosynthesis, is a potential target for AML therapy. AML cells expressing shRNA-PAICS exhibited a proliferative disadvantage, indicating a toxic effect of shRNA-PAICS. Treatment of human AML cells with a PAICS inhibitor suppressed their proliferation by inhibiting DNA synthesis and promoting apoptosis and had anti-leukemic effects in AML PDX models. Furthermore, CRISPR/Cas9 screens using AML cells in the presence of the inhibitor revealed genes mediating resistance or synthetic lethal to PAICS inhibition. Our findings identify PAICS as a novel therapeutic target for AML and further define components of de novo purine synthesis pathway and its downstream effectors essential for AML cell survival.

Rapid technological advances in transcriptomics and lineage tracing technologies provide new opportunities to understand organismal development at the single-cell level. Building on these advances, various computational methods have been proposed to infer developmental trajectories and to predict cell fate. These methods have unveiled previously uncharacterized transitional cell types and differentiation processes. Importantly, the ability to recover cell states and trajectories has been evolving hand-in-hand with new technologies and diverse experimental designs; more recent methods can capture complex trajectory topologies and infer short- and long-term cell fate dynamics. Here, we summarize and categorize the most recent and popular computational approaches for trajectory inference based on the information they leverage and describe future challenges and opportunities for the development of new methods for reconstructing differentiation trajectories and inferring cell fates.

Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are both derived from epidermal keratinocytes but are phenotypically diverse. To improve the understanding of keratinocyte carcinogenesis, it is critical to understand epigenetic alterations, especially those that govern gene expression. We examined changes to the enhancer-associated histone acetylation mark H3K27ac by mapping matched tumor-normal pairs from 11 patients (five with BCC and six with SCC) undergoing Mohs surgery. Our analysis uncovered cancer-specific enhancers on the basis of differential H3K27ac peaks between matched tumor-normal pairs. We also uncovered biological pathways potentially altered in keratinocyte carcinoma, including enriched epidermal development and Wnt signaling pathways enriched in BCCs and enriched immune response and cell activation pathways in SCCs. We also observed enrichment of transcription factors that implicated SMAD and JDP2 in BCC pathogenesis and FOXP1 in SCC pathogenesis. On the basis of these findings, we prioritized three loci with putative regulation events (FGFR2 enhancer in BCC, intragenic regulation of FOXP1 in SCC, and WNT5A promoter in both subtypes) and validated our findings with published gene expression data. Our findings highlight unique and shared epigenetic alterations in histone modifications and potential regulators for BCCs and SCCs that likely impact the divergent oncogenic pathways, paving the way for targeted drug discoveries.

Known fetal hemoglobin (HbF) silencers have potential on-target liabilities for rational β-hemoglobinopathy therapeutic inhibition. Here, through transcription factor (TF) CRISPR screening, we identify zinc-finger protein (ZNF) 410 as an HbF repressor. ZNF410 does not bind directly to the genes encoding γ-globins, but rather its chromatin occupancy is concentrated solely at CHD4, encoding the NuRD nucleosome remodeler, which is itself required for HbF repression. CHD4 has two ZNF410-bound regulatory elements with 27 combined ZNF410 binding motifs constituting unparalleled genomic clusters. These elements completely account for the effects of ZNF410 on fetal globin repression. Knockout of ZNF410 or its mouse homolog Zfp410 reduces CHD4 levels by 60%, enough to substantially de-repress HbF while eluding cellular or organismal toxicity. These studies suggest a potential target for HbF induction for β-hemoglobin disorders with a wide therapeutic index. More broadly, ZNF410 represents a special class of gene regulator, a conserved TF with singular devotion to regulation of a chromatin subcomplex.

BCL11A, the major regulator of fetal hemoglobin (HbF, α2γ2) level, represses γ-globin expression through direct promoter binding in adult erythroid cells in a switch to adult hemoglobin (HbA, α2β2). To uncover how BCL11A initiates repression, we used CRISPR–Cas9, dCas9, dCas9-KRAB and dCas9-VP64 screens to dissect the γ-globin promoters and identified an activator element near the BCL11A-binding site. Using CUT&RUN and base editing, we demonstrate that a proximal CCAAT box is occupied by the activator NF-Y. BCL11A competes with NF-Y binding through steric hindrance to initiate repression. Occupancy of NF-Y is rapidly established following BCL11A depletion, and precedes γ-globin derepression and locus control region (LCR)–globin loop formation. Our findings reveal that the switch from fetal to adult globin gene expression within the >50-kb β-globin gene cluster is initiated by competition between a stage-selective repressor and a ubiquitous activating factor within a remarkably discrete region of the γ-globin promoters. CRISPR–Cas9 tiled screens of the β-globin gene cluster identify an NF-Y bound activator element at the γ-globin promoter. Binding competition by the transcriptional repressor BCL11A leads to NF-Y eviction and a switch from fetal to adult globin gene expression.

The FDA approval of the KRAS G12C inhibitor (G12Ci) sotorasib and the advancement of similar drugs into clinical trials marks a major milestone in treating KRAS G12C non-small cell lung cancer (NSCLC). However, not all patients respond (sotorasib - ORR = 37.1%, adagrasib - 43%, JDQ443 - 35%), motivating preclinical and clinical investigation into mechanisms of intrinsic and acquired resistance. For instance, clinical studies have reported on-target KRAS mutations and preclinical studies have demonstrated mitogen-activated protein kinase (MAPK) feedback reactivation including EGFP, SHP2, and WT RAS signaling. In response to targeted therapies, sub-populations of cells can enter quiescence or specific epigenetic-driven states that confer drug tolerance. However, epigenetic states defining drug-tolerant persister populations and contributing to adaptive resistance to KRAS G12Ci have not been reported. Using a lineage tracing barcoded system, we identify distinct and reversible subpopulations defined by specific chromatin and transcriptional states in KRAS NSCLC cell lines that contribute to KRAS G12Ci resistance in vitro, even prior to drug treatment. We observed that specific states, including activation of histone demethylation and SWI/SNF complex, may contribute to MAPK reactivation-driven resistance. These results suggest potential epigenetic vulnerabilities that can be exploited to improve the response to KRAS G12Ci. Moreover, we observed distinct persister subpopulations with resistance to KRAS G12Ci combination co-targeting orthogonal pathways (SHP2, CDK4/6, PI3K, and MCL-1), raising the possibility that distinct epigenetic-transcriptional states contribute to differential drug response and clonal evolution of persisters. Collectively, these results suggest that more complete tumor regression may be achieved by orthogonal strategies that target different resistant populations within the same tumor. Citation Format: Chendi Li, Qian Qin, Mohammed Usman Syed, Anahita Nimbalkar, Barbara Karakyriakou, Sarah E. Clark, Anne Y. Saiki, Paul E. Hughes, Chris Ott, Luca Pinello, Aaron N. Hata. Chromatin modification driving sub-clonal resistance to KRAS G12C combination therapies in KRAS mutant non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3867.

Prime editing (PE) is a versatile genome editing technology, but design of the required guide RNAs is more complex than for standard CRISPR-based nucleases or base editors. Here we describe PrimeDesign, a user-friendly, end-to-end web application and command-line tool for the design of PE experiments. PrimeDesign can be used for single and combination editing applications, as well as genome-wide and saturation mutagenesis screens. Using PrimeDesign, we construct PrimeVar, a comprehensive and searchable database that includes candidate prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations for installing or correcting >68,500 pathogenic human genetic variants from the ClinVar database. Finally, we use PrimeDesign to design pegRNAs/ngRNAs to install a variety of human pathogenic variants in human cells., Prime editing guide RNA design is more complex than for standard CRISPR-based nucleases or base editors. Here the authors present PrimeDesign and PrimeVar for the rapid and simplified design of pegRNA and ngRNA combinations.

Recent advances in single-cell omics technologies enable the individual and joint profiling of cellular measurements including gene expression, epigenetic features, chromatin structure and DNA sequences. Currently, most single-cell analysis pipelines are cluster-centric, i.e., they first cluster cells into non-overlapping cellular states and then extract their defining genomic features. These approaches assume that discrete clusters correspond to biologically relevant subpopulations and do not explicitly model the interactions between different feature types. In addition, single-cell methods are generally designed for a particular task as distinct single-cell problems are formulated differently. To address these current shortcomings, we present SIMBA, a graph embedding method that jointly embeds single cells and their defining features, such as genes, chromatin accessible regions, and transcription factor binding sequences into a common latent space. By leveraging the co-embedding of cells and features, SIMBA allows for the study of cellular heterogeneity, clustering-free marker discovery, gene regulation inference, batch effect removal, and omics data integration. SIMBA has been extensively applied to scRNA-seq, scATAC-seq, and dual-omics data. We show that SIMBA provides a single framework that allows diverse single-cell analysis problems to be formulated in a unified way and thus simplifies the development of new analyses and integration of other single-cell modalities.

Regulatory elements (REs) consist of enhancers and promoters that occupy a significant portion of the noncoding genome and control gene expression programs either in cis or in trans. Putative REs have been identified largely based on their regulatory features (co-occupancy of ESC-specific transcription factors, enhancer histone marks, and DNase hypersensitivity) in mouse embryonic stem cells (mESCs). However, less has been established regarding their regulatory functions in their native context. We deployed cis- and trans-regulatory elements scanning through saturating mutagenesis and sequencing (ctSCAN-SMS) to target elements within the ∼12-kb cis-region (cis-REs; CREs) of the Oct4 gene locus, as well as genome-wide 2,613 high-confidence trans-REs (TREs), in mESCs. ctSCAN-SMS identified 10 CREs and 12 TREs as novel candidate REs of the Oct4 gene in mESCs. Furthermore, deletions of these candidate REs confirmed that the majority of the REs are functionally active, and CREs are more active than TREs in controlling Oct4 gene expression. A subset of active CREs and TREs physically interact with the Oct4 promoter to varying degrees; specifically, a greater number of active CREs, compared with active TREs, physically interact with the Oct4 promoter. Moreover, comparative genomics analysis reveals that a greater number of active CREs than active TREs are evolutionarily conserved between mice and primates, including humans. Taken together, our study demonstrates the reliability and robustness of ctSCAN-SMS screening to identify critical REs and investigate their roles in the regulation of transcriptional output of a target gene (in this case Oct4) in their native context.

The CRISPR-Cas system offers a programmable platform for eukaryotic genome and epigenome editing. The ability to perform targeted genetic and epigenetic perturbations enables researchers to perform a variety of tasks, ranging from investigating questions in basic biology to potentially developing novel therapeutics for the treatment of disease. While CRISPR systems have been engineered to target DNA and RNA with increased precision, efficiency, and flexibility, assays to identify off-target editing are becoming more comprehensive and sensitive. Furthermore, techniques to perform high-throughput genome and epigenome editing can be paired with a variety of readouts and are uncovering important cellular functions and mechanisms. These technological advances drive and are driven by accompanying computational approaches. Here, we briefly present available CRISPR technologies and review key computational advances and considerations for various CRISPR applications. In particular, we focus on the analysis of on- and off-target editing and CRISPR pooled screen data.

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 BCL11A erythroid 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 BCL11A enhancer 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 BCL11A erythroid 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. A single therapeutic base edit of the BCL11A enhancer in human HSPCs can ameliorate cellular defects associated with sickle cell disease and β-thalassemia in vitro and efficiently induce fetal hemoglobin expression upon engraftment in mice in vivo.

Methods for in vitro DNA cleavage and molecular cloning remain unable to precisely cleave DNA directly adjacent to bases of interest. Restriction enzymes (REs) must bind specific motifs, whereas wild-type CRISPR-Cas9 or CRISPR-Cas12 nucleases require protospacer adjacent motifs (PAMs). Here we explore the utility of our previously reported near-PAMless SpCas9 variant, named SpRY, to serve as a universal DNA cleavage tool for various cloning applications. By performing SpRY DNA digests (SpRYgests) using more than 130 guide RNAs (gRNAs) sampling a wide diversity of PAMs, we discovered that SpRY is PAMless in vitro and can cleave DNA at practically any sequence, including sites refractory to cleavage with wild-type SpCas9. We illustrate the versatility and effectiveness of SpRYgests to improve the precision of several cloning workflows, including those not possible with REs or canonical CRISPR nucleases. We also optimize a rapid and simple one-pot gRNA synthesis protocol to streamline SpRYgest implementation. Together, SpRYgests can improve various DNA engineering applications that benefit from precise DNA breaks.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood and is comprised of at least two major molecular subtypes. Despite sharing features with skeletal muscle, the conservation of underlying cellular hierarchy with human muscle development and the identification of molecularly-defined tumor-propagating cells have not been reported. Using single-cell RNA sequencing of patient-derived RMS, DNA-barcode cell fate mapping, antibody enrichment and functional stem cell assays, and mouse xenograft modeling, we have uncovered tumor cell hierarchies in Fusion-negative (FN-) RMS that are shared with normal human muscle development. We also identified common developmental stages at which tumor cells become arrested. FN-RMS resemble early muscle found in embryonic and larval development, while fusion-positive (FP-) RMS express a highly specific developmental gene program found in muscle cells transiting from embryonic to fetal development at 7-7.75 weeks of age. FP-RMS also have neural-pathway enriched cell states, suggesting less-rigid adherence to muscle development hierarchies in this disease. Finally, we identify a new molecularly-defined tumor-propagating cell in FN-RMS that shares remarkable similarity to the newly described bi-potent, muscle mesenchyme stem/progenitor cell that makes both muscle and osteogenic cells. Citation Format: David M. Langenau, Yun Wei, Qian Qin, Luca Pinello. Stem cell and developmental hierarchies in rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA014.

LSD1 (KDM1A) is a histone demethylase that plays both oncogenic and tumor suppressor roles in breast cancer. However, the exact contexts under which it plays these opposite functions remain largely elusive. By characterizing its role in luminal breast epithelial cells, here we show that inhibition of LSD1 by both genetic and pharmacological approaches increases their invasion and migration, whereas its inhibition by genetic approach, but not by pharmacological approach, impairs their proliferation/survival. Induced loss of LSD1 in luminal cells in a mouse model of luminal breast cancer, MMTV-PyMT, leads to a profound increase in lung metastasis. Mechanistically, LSD1 interacts with GATA3, a key luminal-specific transcription factor (TF), and their common target genes are highly related to breast cancer. LSD1 positively regulates GATA3 expression. It also represses expression of TRIM37, a breast epithelial oncogene encoding a histone H2A ubiquitin ligase, and ELF5, a key TF gene for luminal progenitors and alveolar luminal cells. LSD1-loss also leads to reduced expression of several cell-cell adhesion genes (e.g., CDH1, VCL, CTNNA1), possibly via TRIM37-upregulation and subsequently TRIM37-mediated repression. Collectively, our data suggest LSD1 largely plays a tumor suppressor role in luminal breast cancer and the oncogenic program associated with LSD1-inhibition may be suppressed via TRIM37-inhibition.

Single-cell transcriptomic assays have enabled the de novo reconstruction of lineage differentiation trajectories, along with the characterization of cellular heterogeneity and state transitions. Several methods have been developed for reconstructing developmental trajectories from single-cell transcriptomic data, but efforts on analyzing single-cell epigenomic data and on trajectory visualization remain limited. Here we present STREAM, an interactive pipeline capable of disentangling and visualizing complex branching trajectories from both single-cell transcriptomic and epigenomic data. We have tested STREAM on several synthetic and real datasets generated with different single-cell technologies. We further demonstrate its utility for understanding myoblast differentiation and disentangling known heterogeneity in hematopoiesis for different organisms. STREAM is an open-source software package., The increasing accessibility of single cell omics technologies beyond transcriptomics demands parallel advances in analysis. Here, the authors introduce STREAM, a pipeline for reconstruction and visualization of differentiation trajectories from both single-cell RNA-seq and ATAC-seq data.

Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases1 has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM)2. To address this limitation, we engineered an enhanced Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range, enabling targeting of many previously inaccessible PAMs. On average, enAsCas12a exhibits two-fold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a, and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants3 to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing, endogenous gene activation, and C-to-T base editing, and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively, enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing. [AU: Revised abstract OK?]

CRISPR gene editing holds great promise to modify somatic genomes to ameliorate disease. In silico prediction of homologous sites coupled with biochemical evaluation of possible genomic off-targets may predict genotoxicity risk of individual gene editing reagents. However, standard computational and biochemical methods focus on reference genomes and do not consider the impact of genetic diversity on off-target potential. Here we developed a web application called CRISPRme that explicitly and efficiently integrates human genetic variant datasets with orthogonal genomic annotations to nominate and prioritize off-target sites at scale. The method considers both single-nucleotide variants (SNVs) and indels, accounts for bona fide haplotypes, accepts spacer:protospacer mismatches and bulges, and is suitable for personal genome analyses. We tested the tool with a guide RNA (gRNA) targeting the BCL11A erythroid enhancer that has shown therapeutic promise in clinical trials for sickle cell disease (SCD) and β-thalassemia1. We find that the top candidate off-target site is produced by a non-reference allele common in African-ancestry populations (rs114518452, minor allele frequency (MAF)=4.5%) that introduces a protospacer adjacent motif (PAM) for SpCas9. We validate that SpCas9 generates indels (∼9.6% frequency) and chr2 pericentric inversions in a strictly allele-specific manner in edited CD34+ hematopoietic stem/progenitor cells (HSPCs), although a high-fidelity Cas9 variant mitigates this off-target. The CRISPRme tool highlights alternative allele-specific off-target editing as a prevalent risk of gRNAs considered for therapeutic gene editing. Our report illustrates how population and private genetic variants should be considered as modifiers of genome editing outcomes. We suggest that variant-aware off-target assessment should be considered in therapeutic genome editing efforts going forward and provide a powerful approach for comprehensive off-target nomination.