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Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos.
Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single-nucleotide polymorphism in individuals. Here we developed a method named GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole-genome sequences of progeny cells of edited and nonedited blastomeres at embryonic day 14.5 showed that off-target single-nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. By contrast, cytosine base editing induced SNVs at more than 20-fold higher frequencies, requiring a solution to address its fidelity.
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Transcription-wide impact by RESCUE-induced off-target single-nucleotide variants in mammalian cells.
RNA base editing is a promising tool in precise molecular therapy. Currently, there are two widely used RNA base editors, REPAIR and RESCUE. REPAIR only facilitates A-to-I conversions, while RESCUE performs both A-to-I and C-to-U conversions. Thus, RESCUE can generate twice the number of mutations compared to REPAIR. However, transcription-wide impact due to RESCUE-induced off-target single-nucleotide variants (SNVs) is not fully appreciated. Therefore, to determine the off-target effects of RESCUE-mediated editing, we employed transcription-wide sequencing on cells edited by RESCUE. The SNVs showed different off-target effects on mRNA, circRNA, lncRNA, and miRNA expression patterns and their interacting networks. Our results illustrate the transcription-wide impact of RESCUE-induced off-target SNVs and highlight the need for careful characterization of the off-target impact by this editing platform.
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Assessing and engineering the IscB-ωRNA system for programmed genome editing.
OMEGA RNA (ωRNA)-guided endonuclease IscB, the evolutionary ancestor of Cas9, is an attractive system for in vivo genome editing because of its compact size and mechanistic resemblance to Cas9. However, wild-type IscB-ωRNA systems show limited activity in human cells. Here we report enhanced OgeuIscB, which, with eight amino acid substitutions, displayed a fourfold increase in in vitro DNA-binding affinity and a 30.4-fold improvement in insertion-deletion (indel) formation efficiency in human cells. Paired with structure-guided ωRNA engineering, the enhanced OgeuIscB-ωRNA systems efficiently edited the human genome across 26 target sites, attaining up to 87.3% indel and 62.2% base-editing frequencies. Both wild-type and engineered OgeuIscB-ωRNA showed moderate fidelity in editing the human genome, with off-target profiles revealing key determinants of target selection including an NARR target-adjacent motif (TAM) and the TAM-proximal 14 nucleotides in the R-loop. Collectively, our engineered OgeuIscB-ωRNA systems are programmable, potent and sufficiently specific for human genome editing.
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Clinical development of allogeneic chimeric antigen receptor αβ-T cells.
Ready-made banks of allogeneic chimeric antigen receptor (CAR) T cells, produced to be available at the time of need, offer the prospect of accessible and cost-effective cellular therapies. Various strategies have been developed to overcome allogeneic barriers, drawing on cell engineering platforms including RNA interference, protein-based restriction, and genome editing, including RNA-guided CRISPR-Cas and base editing tools. Alloreactivity and the risk of graft-versus-host disease from non-matched donor cells have been mitigated by disruption of αβ-T cell receptor expression on the surface of T cells and stringent removal of any residual αβ-T cell populations. In addition, host-mediated rejection has been tackled through a combination of augmented lymphodepletion and cell engineering strategies that have allowed infused cells to evade immune recognition or conferred resistance to lymphodepleting agents to promote persistence and expansion of effector populations. Early-phase studies using off-the-shelf universal donor CAR T cells have been undertaken mainly in the context of blood malignancies, where emerging data of clinical responses have supported wider adoption and further applications. These developments offer the prospect of alternatives to current autologous approaches through the emerging application of genome engineering solutions to improve safety, persistence, and function of universal donor products.
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Base editing of haematopoietic stem cells rescues sickle cell disease in mice.
Sickle cell disease (SCD) is caused by a mutation in the β-globin gene HBB1. We used a custom adenine base editor (ABE8e-NRCH)2,3 to convert the SCD allele (HBBS) into Makassar β-globin (HBBG), a non-pathogenic variant4,5. Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBBS to HBBG. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBBG was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBBS base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse6 and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar β-globin represented 79% of β-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBBS-to-HBBG editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBBS, generates benign HBBG, and minimizes the undesired consequences of double-strand DNA breaks.
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Cooperativity between Cas9 and hyperactive AID establishes broad and diversifying mutational footprints in base editors.
The partnership of DNA deaminase enzymes with CRISPR-Cas nucleases is now a well-established method to enable targeted genomic base editing. However, an understanding of how Cas9 and DNA deaminases collaborate to shape base editor (BE) outcomes has been lacking. Here, we support a novel mechanistic model of base editing by deriving a range of hyperactive activation-induced deaminase (AID) base editors (hBEs) and exploiting their characteristic diversifying activity. Our model involves multiple layers of previously underappreciated cooperativity in BE steps including: (i) Cas9 binding can potentially expose both DNA strands for 'capture' by the deaminase, a feature that is enhanced by guide RNA mismatches; (ii) after strand capture, the intrinsic activity of the DNA deaminase can tune window size and base editing efficiency; (iii) Cas9 defines the boundaries of editing on each strand, with deamination blocked by Cas9 binding to either the PAM or the protospacer and (iv) non-canonical edits on the guide RNA bound strand can be further elicited by changing which strand is nicked by Cas9. Leveraging insights from our mechanistic model, we create novel hBEs that can remarkably generate simultaneous C > T and G > A transitions over >65 bp with significant potential for targeted gene diversification.
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Discriminated sgRNAs-Based SurroGate System Greatly Enhances the Screening Efficiency of Plant Base-Edited Cells.
The development of CRISPR/Cas9-mediated base editing has made genomic modification more efficient. However, selection of genetically modified cells from millions of treated cells, especially plant cells, is still challenging. In this study, an efficient surrogate reporter system based on a defective hygromycin resistance gene was established in rice to enrich base-edited cells. After step-by-step optimization, the Discriminated sgRNAs-based SurroGate system (DisSUGs) was established by artificially differentiating the editing abilities of a wild-type single guide RNA (sgRNA) targeting the surrogate reporter gene and an enhanced sgRNA targeting endogenous sites. The DisSUGs enhanced the efficiency of screening base-edited cells by 3- to 5-fold for a PmCDA1-based cytosine-to-tyrosine base editor (PCBE), and 2.5- to 6.5-fold for an adenine base editor (ABE) at endogenous targets. These targets showed editing efficiencies of <25% in the conventional systems. The DisSUGs greatly enhanced the frequency of homozygous substitutions and expanded the activity window slightly for both a PCBE and an ABE. Analyses of the total number of single-nucleotide variants from whole-genome sequencing revealed that, compared with the no-enrichment PCBE strategy, the DisSUGs did not alter the frequency of genome-wide sgRNA-independent off-target mutations, but slightly increased the frequency of target-dependent off-target mutations. Collectively, the DisSUGs developed in this study greatly enhances the efficiency of screening plant base-edited cells and will be a useful system in future applications.
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Simultaneous targeting of linked loci in mouse embryos using base editing.
A particular challenge in genome engineering has been the simultaneous introduction of mutations into linked (located on the same chromosome) loci. Although CRISPR/Cas9 has been widely used to mutate individual sites, its application in simultaneously targeting of linked loci is limited as multiple nearby double-stranded DNA breaks created by Cas9 routinely result in the deletion of sequences between the cleavage sites. Base editing is a newer form of genome editing that directly converts C∙G-to-T∙A, or A∙T-to-G∙C, base pairs without introducing double-stranded breaks, thus opening the possibility to generate linked mutations without disrupting the entire locus. Through the co-injection of two base editors and two sgRNAs into mouse zygotes, we introduced C∙G-to-T∙A transitions into two cytokine-sensing transcription factor binding sites separated by 9 kb. We determined that one enhancer activates the two flanking genes in mammary tissue during pregnancy and lactation. The ability to introduce linked mutations simultaneously in one step into the mammalian germline has implications for a wide range of applications, including the functional analysis of linked cis-elements creating disease models and correcting pathogenic mutations.
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Optimized base editors enable efficient editing in cells, organoids and mice.
CRISPR base editing enables the creation of targeted single-base conversions without generating double-stranded breaks. However, the efficiency of current base editors is very low in many cell types. We reengineered the sequences of BE3, BE4Gam, and xBE3 by codon optimization and incorporation of additional nuclear-localization sequences. Our collection of optimized constitutive and inducible base-editing vector systems dramatically improves the efficiency by which single-nucleotide variants can be created. The reengineered base editors enable target modification in a wide range of mouse and human cell lines, and intestinal organoids. We also show that the optimized base editors mediate efficient in vivo somatic editing in the liver in adult mice.
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Base Editors: Modular Tools for the Introduction of Point Mutations in Living Cells.
Base editors are a new family of programmable genome editing tools that fuse ssDNA (single stranded DNA) modifying enzymes to catalytically inactive CRISPR-associated (Cas) endonucleases to induce highly efficient single base changes. With dozens of base editors now reported, it is apparent that these tools are highly modular; many combinations of ssDNA modifying enzymes and Cas proteins have resulted in a variety of base editors, each with its own unique properties and potential uses. In this perspective, we describe currently available base editors, highlighting their modular nature and describing the various options available for each component. Furthermore, we briefly discuss applications in synthetic biology and genome engineering where base editors have presented unique advantages over alternative techniques.
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