In vivo evaluation of guide-free Cas9-induced safety risks in a pig model.

The CRISPR/Cas9 system has shown great potential for treating human genetic diseases through gene therapy. However, there are concerns about the safety of this system, specifically related to the use of guide-free Cas9. Previous studies have shown that guide-free Cas9 can induce genomic instability in vitro. However, the in vivo safety risks associated with guide-free Cas9 have not been evaluated, which is necessary for the development of gene therapy in clinical settings. In this study, we used doxycycline-inducible Cas9-expressing pigs to evaluate the safety risks of guide-free Cas9 in vivo. Our findings demonstrated that expression of guide-free Cas9 could induce genomic damages and transcriptome changes in vivo. The severity of the genomic damages and transcriptome changes were correlate with the expression levels of Cas9 protein. Moreover, prolonged expression of Cas9 in pigs led to abnormal phenotypes, including a significant decrease in body weight, which may be attributable to genomic damage-induced nutritional absorption and metabolic dysfunction. Furthermore, we observed an increase in whole-genome and tumor driver gene mutations in pigs with long-term Cas9 expression, raising the risk of tumor occurrence. Our in vivo evaluation of guide-free Cas9 in pigs highlights the necessity of considering and monitoring the detrimental effects of Cas9 alone as genome editing via the CRISPR/Cas9 system is implemented in clinical gene therapy. This research emphasizes the importance of further study and implementation of safety measures to ensure the successful and safe application of the CRISPR/Cas9 system in clinical practice.

Improvement of TaC9-ABE mediated correction of human SMN2 gene.

Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by mutations in the survival motor neuron 1 (SMN1) gene. Gene editing technology repairs the conversion of the 6th base T to C in exon 7 of the paralogous SMN2 gene, compensating for the SMN protein expression and promoting the survival and function of motor neurons. However, low editing efficiency and unintended off-target effects limit the application of this technology. Here, we optimized a TaC9-adenine base editor (ABE) system by combining Cas9 nickase with the transcription activator-like effector (TALE)-adenosine deaminase fusion protein to effectively and precisely edit SMN2 without detectable Cas9 dependent off-target effects in human cell lines. We also generated human SMA-induced pluripotent stem cells (SMA-iPSCs) through the mutation of the splice acceptor or deletion of the exon 7 of SMN1. TaC9-R10 induced 45% SMN2 T6 > C conversion in the SMA-iPSCs. The SMN2 T6 > C splice-corrected SMA-iPSCs were directionally differentiated into motor neurons, exhibiting SMN protein recovery and antiapoptosis ability. Therefore, the TaC9-ABE system with dual guides from the combination of Cas9 with TALE could be a potential therapeutic strategy for SMA with high efficacy and safety.

Enhancing prime editor flexibility with coiled-coil heterodimers.

BACKGROUND: Prime editing enables precise base substitutions, insertions, and deletions at targeted sites without the involvement of double-strand DNA breaks or exogenous donor DNA templates. However, the large size of prime editors (PEs) hampers their delivery in vivo via adeno-associated virus (AAV) due to the viral packaging limit. Previously reported split PE versions provide a size reduction, but they require intricate engineering and potentially compromise editing efficiency. RESULTS: Herein, we present a simplified split PE named as CC-PE, created through non-covalent recruitment of reverse transcriptase to the Cas9 nickase via coiled-coil heterodimers, which are widely used in protein design due to their modularity and well-understood sequence-structure relationship. We demonstrate that the CC-PE maintains or even surpasses the efficiency of unsplit PE in installing intended edits, with no increase in the levels of undesired byproducts within tested loci amongst a variety of cell types (HEK293T, A549, HCT116, and U2OS). Furthermore, coiled-coil heterodimers are used to engineer SpCas9-NG-PE and SpRY-PE, two Cas9 variants with more flexible editing scope. Similarly, the resulting NG-CC-PE and SpRY-CC-PE also achieve equivalent or enhanced efficiency of precise editing compared to the intact PE. When the dual AAV vectors carrying CC-PE are delivered into mice to target the Pcsk9 gene in the liver, CC-PE enables highly efficient precise editing, resulting in a significant reduction of plasma low-density lipoprotein cholesterol and total cholesterol. CONCLUSIONS: Our innovative, modular system enhances flexibility, thus potentially facilitating the in vivo applicability of prime editing.

An optimized culture system for efficient derivation of porcine expanded potential stem cells from preimplantation embryos and by reprogramming somatic cells.

Pigs share anatomical and physiological traits with humans and can serve as a large-animal model for translational medicine. Bona fide porcine pluripotent stem cells (PSCs) could facilitate testing cell and drug therapies. Agriculture and biotechnology may benefit from the ability to produce immune cells for studying animal infectious diseases and to readily edit the porcine genome in stem cells. Isolating porcine PSCs from preimplantation embryos has been intensively attempted over the past decades. We previously reported the derivation of expanded potential stem cells (EPSCs) from preimplantation embryos and by reprogramming somatic cells of multiple mammalian species, including pigs. Porcine EPSCs (pEPSCs) self-renew indefinitely, differentiate into embryonic and extra-embryonic lineages, and permit precision genome editing. Here we present a highly reproducible experimental procedure and data of an optimized and robust porcine EPSC culture system and its use in deriving new pEPSC lines from preimplantation embryos and reprogrammed somatic cells. No particular expertise is required for the protocols, which take ~4-6 weeks to complete. Importantly, we successfully established pEPSC lines from both in vitro fertilized and somatic cell nuclear transfer-derived embryos. These new pEPSC lines proliferated robustly over long-term passaging and were amenable to both simple indels and precision genome editing, with up to 100% targeting efficiency. The pEPSCs differentiated into embryonic cell lineages in vitro and teratomas in vivo, and into porcine trophoblast stem cells in human trophoblast stem cell medium. We show here that pEPSCs have unique epigenetic features, particularly H3K27me3 levels substantially lower than fibroblasts.

A strategy for Cas13 miniaturization based on the structure and AlphaFold.

The small size of the Cas nuclease fused with various effector domains enables a broad range of function. Although there are several ways of reducing the size of the Cas nuclease complex, no efficient or generalizable method has been demonstrated to achieve protein miniaturization. In this study, we establish an Interaction, Dynamics and Conservation (IDC) strategy for protein miniaturization and generate five compact variants of Cas13 with full RNA binding and cleavage activity comparable the wild-type enzymes based on a combination of IDC strategy and AlphaFold2. In addition, we construct an RNA base editor, mini-Vx, and a single AAV (adeno-associated virus) carrying a mini-RfxCas13d and crRNA expression cassette, which individually shows efficient conversion rate and RNA-knockdown activity. In summary, these findings highlight a feasible strategy for generating downsized CRISPR/Cas13 systems based on structure predicted by AlphaFold2, enabling targeted degradation of RNAs and RNA editing for basic research and therapeutic applications.

VCFshiny: an R/Shiny application for interactively analyzing and visualizing genetic variants.

Summary: Next-generation sequencing generates variants that are typically documented in variant call format (VCF) files. However, comprehensively examining variant information from VCF files can pose a significant challenge for researchers lacking bioinformatics and programming expertise. To address this issue, we introduce VCFshiny, an R package that features a user-friendly web interface enabling interactive annotation, interpretation, and visualization of variant information stored in VCF files. VCFshiny offers two annotation methods, Annovar and VariantAnnotation, to add annotations such as genes or functional impact. Annotated VCF files are deemed acceptable inputs for the purpose of summarizing and visualizing variant information. This includes the total number of variants, overlaps across sample replicates, base alterations of single nucleotides, length distributions of insertions and deletions (indels), high-frequency mutated genes, variant distribution in the genome and of genome features, variants in cancer driver genes, and cancer mutational signatures. VCFshiny serves to enhance the intelligibility of VCF files by offering an interactive web interface for analysis and visualization. Availability and implementation: The source code is available under an MIT open source license at https://github.com/123xiaochen/VCFshiny with documentation at https://123xiaochen.github.io/VCFshiny.

Mini-PE, a prime editor with compact Cas9 and truncated reverse transcriptase.

Prime editor (PE) is a versatile genome editing tool that does not need extra DNA donors or inducing double-strand breaks. However, in vivo implementation of PE remains a challenge because of its oversized composition. In this study, we screened out the smallest truncated Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) with the F155Y mutation to keep gene editing efficiency. We discovered the most efficient gene editing variants of MMLV RT with the smallest size. After optimization of the pegRNAs and incorporation with nick sgRNAs, the mini-PE delivered up to 10% precise editing at target sites in human and mouse cells. It also edited the mouse Hsf1 gene in the mouse retina precisely after delivery with adeno-associated viruses (AAVs), although the editing efficiency was lower than 1%. We will focus on improving the editing efficiency of mini-PE and exploiting its therapeutic potential against human genetic diseases.

BCL11B and the NuRD complex cooperatively guard T-cell fate and inhibit OPA1-mediated mitochondrial fusion in T cells.

The nucleosome remodeling and histone deacetylase (NuRD) complex physically associates with BCL11B to regulate murine T-cell development. However, the function of NuRD complex in mature T cells remains unclear. Here, we characterize the fate and metabolism of human T cells in which key subunits of the NuRD complex or BCL11B are ablated. BCL11B and the NuRD complex bind to each other and repress natural killer (NK)-cell fate in T cells. In addition, T cells upregulate the NK cell-associated receptors and transcription factors, lyse NK-cell targets, and are reprogrammed into NK-like cells (ITNKs) upon deletion of MTA2, MBD2, CHD4, or BCL11B. ITNKs increase OPA1 expression and exhibit characteristically elongated mitochondria with augmented oxidative phosphorylation (OXPHOS) activity. OPA1-mediated elevated OXPHOS enhances cellular acetyl-CoA levels, thereby promoting the reprogramming efficiency and antitumor effects of ITNKs via regulating H3K27 acetylation at specific targets. In conclusion, our findings demonstrate that the NuRD complex and BCL11B cooperatively maintain T-cell fate directly by repressing NK cell-associated transcription and indirectly through a metabolic-epigenetic axis, providing strategies to improve the reprogramming efficiency and antitumor effects of ITNKs.

Generation of a humanized mesonephros in pigs from induced pluripotent stem cells via embryo complementation.

Heterologous organ transplantation is an effective way of replacing organ function but is limited by severe organ shortage. Although generating human organs in other large mammals through embryo complementation would be a groundbreaking solution, it faces many challenges, especially the poor integration of human cells into the recipient tissues. To produce human cells with superior intra-niche competitiveness, we combined optimized pluripotent stem cell culture conditions with the inducible overexpression of two pro-survival genes (MYCN and BCL2). The resulting cells had substantially enhanced viability in the xeno-environment of interspecies chimeric blastocyst and successfully formed organized human-pig chimeric middle-stage kidney (mesonephros) structures up to embryonic day 28 inside nephric-defective pig embryos lacking SIX1 and SALL1. Our findings demonstrate proof of principle of the possibility of generating a humanized primordial organ in organogenesis-disabled pigs, opening an exciting avenue for regenerative medicine and an artificial window for studying human kidney development.

Generation of hiPSCs with ABO c.767T>C substitution: resulting in splicing variants.

Introduction: The ABO blood group system has important clinical significance in the safety of blood transfusion and organ transplantation. Numerous ABO variations, especially variations in the splice sites, have been identified to be associated with some ABO subtypes. Methods: Here, we performed the c.767T>C substitution of the ABO gene in human induced pluripotent stem cells (hiPSCs) by the adenosine base editor (ABE) system and described its characteristics at the genome level in detail. Results: The hiPS cell line with c.767T>C substitution maintained a normal karyotype (46, XX), expressed pluripotency markers, and showed the capability to spontaneously differentiate into all three germ layers in vivo. The genome-wide analysis demonstrated that the c.767T>C substitution in the ABO gene did not cause any detected negative effect in hiPSCs at the genome level. The splicing transcript analysis revealed that splicing variants were observed in the hiPSCs with ABO c.767T>C substitutions. Conclusion: All these results indicated that some splicing variants occurred in hiPSCs with c.767 T>C substitution of ABO gene, which probably had a significant effect on the formation of the rare ABO*Ael05/B101 subtype.

A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9.

BACKGROUND: Adenine base editors (ABEs) are promising therapeutic gene editing tools that can efficiently convert targeted A•T to G•C base pairs in the genome. However, the large size of commonly used ABEs based on SpCas9 hinders its delivery in vivo using certain vectors such as adeno-associated virus (AAV) during preclinical applications. Despite a number of approaches having previously been attempted to overcome that challenge, including split Cas9-derived and numerous domain-deleted versions of editors, whether base editor (BE) and prime editor (PE) systems can also allow deletion of those domains remains to be proven. In this study, we present a new small ABE (sABE) with significantly reduced size. RESULTS: We discovered that ABE8e can tolerate large single deletions in the REC2 (Δ174-296) and HNH (Δ786-855) domains of SpCas9, and these deletions can be stacked together to create a new sABE. The sABE showed higher precision than the original ABE8e, with proximally shifted protospacer adjacent motif (PAM) editing windows (A3- A15), and comparable editing efficiencies to 8e-SaCas9-KKH. The sABE system efficiently generated A-G mutations at disease-relevant loci (T1214C in GAA and A494G in MFN2) in HEK293T cells and several canonical Pcsk9 splice sites in N2a cells. Moreover, the sABE enabled in vivo delivery in a single adeno-associated virus (AAV) vector with slight efficiency. Furthermore, we also successfully edited the genome of mouse embryos by microinjecting mRNA and sgRNA of sABE system into zygotes. CONCLUSIONS: We have developed a substantially smaller sABE system that expands the targeting scope and offers higher precision of genome editing. Our findings suggest that the sABE system holds great therapeutic potential in preclinical applications.

A new vaccination regimen using adenovirus-vectored vaccine confers effective protection against African swine fever virus in swine.

African swine fever (ASF) is an acute and highly contagious lethal infectious disease in swine that severely threatens the global pig industry. At present, a safe and efficacious vaccine is urgently required to prevent and control the disease. In this study, we evaluated the safety and immunogenicity of replication-incompetent type-2 adenoviruses carrying African swine fever virus (ASFV) antigens, namely CP204L (p30), E183L (p54), EP402R (CD2v), B646L (p72), and B602L (p72 chaperone). A vaccine cocktail delivered by simultaneous intramuscular (IM) and intranasal (IN) administration robustly elicited both systemic and mucosal immune responses against AFSV in mice and swine and provided highly effective protection against the circulating ASFV strain in farmed pigs. This multi-antigen cocktail vaccine was well tolerated in the vaccinated animals. No significant interference among antigens was observed. The combined IM and IN vaccination using this adenovirus-vectored antigen cocktail vaccine warrants further evaluation for providing safe and effective protection against ASFV infection and transmission.

Two-dimensional peptide nanosheets functionalized with gold nanorods for photothermal therapy of tumors.

Self-assembled peptide nanomaterials exhibit great potential for applications in materials science, energy storage, nanodevices, analytical science, biomedicine, tissue engineering, and others due to their tailorable ordered nanostructures and unique physical, chemical, and biological properties. Although one-dimensional peptide nanofibers and nanotubes have been widely used for biomedical applications, the design and synthesis of two-dimensional (2D) peptide nanostructures for cancer therapy remain challenging. In this work, we describe the creation of 2D biocompatible peptide nanosheets (PNSs) through molecular self-assembly, which can provide support matrixes for conjugating gold nanorods (AuNRs) to form high-performance 2D nanomaterials for photothermal conversion. After molecular modification, AuNRs can be chemically conjugated onto the surface of 2D PNSs, and the created PNS-AuNR nanohybrids serve as a potential nanoplatform for photothermal therapy of tumor cells. The obtained results indicate that both PNSs and AuNRs contribute to the improved efficiency of photothermal therapy (PTT) of tumors, in which 2D PNSs provide high biocompatibility and a large surface area for binding AuNRs, and AuNRs show a high PTT ability towards tumors. The strategies of molecular design and functional tailoring of self-assembled peptide nanomaterials shown in this study are valuable and inspire the synthesis of biomimetic nanomaterials for biomedicine and tissue engineering applications.

Cas9-mediated replacement of expanded CAG repeats in a pig model of Huntington's disease.

The monogenic nature of Huntington's disease (HD) and other neurodegenerative diseases caused by the expansion of glutamine-encoding CAG repeats makes them particularly amenable to gene therapy. Here we show the feasibility of replacing expanded CAG repeats in the mutant HTT allele with a normal CAG repeat in genetically engineered pigs mimicking the selective neurodegeneration seen in patients with HD. A single intracranial or intravenous injection of adeno-associated virus encoding for Cas9, a single-guide RNA targeting the HTT gene, and donor DNA containing the normal CAG repeat led to the depletion of mutant HTT in the animals and to substantial reductions in the dysregulated expression and neurotoxicity of mutant HTT and in neurological symptoms. Our findings support the further translational development of virally delivered Cas9-based gene therapies for the treatment of genetic neurodegenerative diseases.

Doxycycline-dependent Cas9-expressing pig resources for conditional in vivo gene nullification and activation.

BACKGROUND: CRISPR-based toolkits have dramatically increased the ease of genome and epigenome editing. SpCas9 is the most widely used nuclease. However, the difficulty of delivering SpCas9 and inability to modulate its expression in vivo hinder its widespread adoption in large animals. RESULTS: Here, to circumvent these obstacles, a doxycycline-inducible SpCas9-expressing (DIC) pig model was generated by precise knock-in of the binary tetracycline-inducible expression elements into the Rosa26 and Hipp11 loci, respectively. With this pig model, in vivo and/or in vitro genome and epigenome editing could be easily realized. On the basis of the DIC system, a convenient Cas9-based conditional knockout strategy was devised through controlling the expression of rtTA component by tissue-specific promoter, which allows the one-step generation of germline-inherited pigs enabling in vivo spatiotemporal control of gene function under simple chemical induction. To validate the feasibility of in vivo gene mutation with DIC pigs, primary and metastatic pancreatic ductal adenocarcinoma was developed by delivering a single AAV6 vector containing TP53-sgRNA, LKB1-sgRNA, and mutant human KRAS gene into the adult pancreases. CONCLUSIONS: Together, these results suggest that DIC pig resources will provide a powerful tool for conditional in vivo genome and epigenome modification for fundamental and applied research.

Repair of bone defects in rhesus monkeys with α1,3-galactosyltransferase-knockout pig cancellous bone.

Introduction: Since xenografts offer a wide range of incomparable advantages, they can be a better option than allografts but only if the possibility of immunological rejection can be eliminated. In this study, we investigated the ability of α1,3-galactosyltransferase (α1,3-GT) gene knockout (GTKO) pig cancellous bone to promote the repair of a femoral condyle bone defect and its influence on heterologous immune rejection. Materials and methods: Cylindrical bone defects created in a rhesus monkey model were transplanted with GTKO bone, WT bone or left empty. For immunological evaluation, T lymphocyte subsets CD4+ and CD8+ in peripheral blood were assayed by flow cytometry, and the IL-2 and IFN-γ contents of peripheral blood serum were analyzed by ELISA at 2, 5, 7, 10, and 14 days post-surgery. Micro-CT scans and histological assessment were conducted at 4 and 8 weeks after implantation. Results: Compared with WT-pig bone, the heterologous immunogenicity of GTKO-pig bone was reduced. The defect filled with fresh GTKO-pig bone was tightly integrated with the graft. Histological analysis showed that GTKO-pig cancellous bone showed better osseointegration and an appropriate rate of resorption. Osteoblast phenotype progression in the GTKO group was not affected, which revealed that GTKO-pig bone could not only fill and maintain the bone defect, but also promote new bone formation. Conclusion: GTKO-pig cancellous bone decreased the ratio of CD4+ to CD8+ T cells and cytokines (IFN-γ and IL-2) to inhibit xenotransplant rejection. Moreover, GTKO group increased more bone formation by micro-CT analysis and osteoblastic markers (Runx2, OSX and OCN). Together, GTKO-pig cancellous bone showed better bone repair than WT-pig cancellous bone.

Inducible motor neuron differentiation of human induced pluripotent stem cells in vivo.

OBJECTIVES: Transplantation of neural progenitor cells (NPCs) derived from human-induced pluripotent stem cells (hiPSCs) is one of the promising treatment strategies for motor neuron diseases (MNDs). However, the inefficiency in committed differentiation of NPCs in vivo limits its application. Here, we tried to establish a potential therapeutic strategy for MNDs by in vivo directional differentiation of hiPSCs engineered with motor neuron (MN) specific transcription factors and Tet-On system. MATERIALS AND METHODS: We engineered hiPSCs with three MN-specific transcription factors and Tet-On system. The engineered cells were directly transplanted into immunodeficient mice through subcutaneous, intra-spinal cord and intracerebroventricular injections. Following doxycycline (Dox) induction, teratoma formation, and motor MN differentiation were evaluated. RESULTS: We generated genetically engineered hiPSCs, in which the expression of Ngn2, Isl1, and Lhx3 was controlled by a drug-inducible transgenic system. These cells showed normal pluripotency and proliferative capacity, and were able to directionally differentiate into mature motor neurons (MNs) and NPCs with high efficiency in spinal cords and cerebral lateral ventricles under the induction of Dox. The grafts showed long-term survival in the recipient mice without formation of teratoma. CONCLUSIONS: The induced mature MNs and NPCs were expected to replace the damaged endogenous MNs directly, and play a role of de novo stem cell stock for long-term neuron damage repair, respectively. Therefore, in vivo directional differentiation of the hiPSCs engineered with MN-specific transcription factors and Tet-On system via Dox induction could be a potential therapeutic strategy for MNDs with high efficacy and safety.

Compact Cje3Cas9 for Efficient In Vivo Genome Editing and Adenine Base Editing.

Many therapeutic applications of CRISPR-Cas9 gene editing rely on delivery using the highly versatile adeno-associated virus (AAV) vector. The smallest type II Cas9 ortholog-Cje1Cas9, derived from Campylobacter jejuni with <1,000 amino acids-is particularly attractive for AAV delivery. However, the complex protospacer adjacent motif (PAM) of Cje1Cas9 (N3VRYAC) greatly restricts the density of recognition sequences in human genome. In this study, we identify two compact CjeCas9 orthologs designated as Cje2Cas9 and Cje3Cas9, whose PAM-interacting residues are different from those of the well-known Cje1Cas9. They can induce efficient genome editing in human cells, and their simpler trinucleotide PAM (N4CYA) requirements expand the scope of targeting. Moreover, Cje3Cas9 efficiently disrupts the Tyr gene in mice after being micro-injected into zygotes with the corresponding sgRNA. It also successfully disrupts the Pcsk9 gene in 8-week-old mouse liver after delivery with an sgRNA using an all-in-one AAV delivery vehicle. The gene-edited mice showed lower cholesterol level than wild-type mice. Notably, the 8e-nCje3-ABE and an sgRNA targeting Pcsk9 were successfully packaged into a single AAV vector for genome editing in adult mouse liver, with editing efficiency up to 12%. Thus, simple PAMs and a compact size enable Cje2/3Cas9 to expand the target scope of CRISPR-Cas9 toolsets, exhibiting considerable potential for therapeutic applications.

Double knock-in pig models with elements of binary Tet-On and phiC31 integrase systems for controllable and switchable gene expression.

Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process. Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline. Previous pig models with tetracycline regulatory elements were generated through random integration. This process often resulted in uncertain expression and unpredictable phenotypes, thus hindering their applications. Here, by precise knock-in of binary Tet-On 3G elements into Rosa26 and Hipp11 locus, respectively, a double knock-in reporter pig model was generated. We characterized excellent properties of this system for controllable transgenic expression both in vitro and in vivo. Two attP sites were arranged to flank the tdTomato to switch reporter gene. Single or multiple gene replacement was efficiently and faithfully achieved in fetal fibroblasts and nuclear transfer embryos. To display the flexible application of this system, we generated a pig strain with Dox-inducing hKRASG12D expression through phiC31 integrase-mediated cassette exchange. After eight months of Dox administration, squamous cell carcinoma developed in the nose, mouth, and scrotum, which indicated this pig strain could serve as an ideal large animal model to study tumorigenesis. Overall, the established pig models with controllable and switchable transgene expression system will provide a facilitating platform for transgenic and biomedical research.

Efficient multinucleotide deletions using deaminase-Cas9 fusions in human cells.

CRISPR/Cas9 system is a robust genome editing platform in biotechnology and medicine. However, it generally produces small insertions/deletions (indels, typically 1-3 bp) but rarely induces larger deletions in specific target sites. Here, we report a cytidine deaminase-Cas9 fusion-induced deletion system (C-DEL) and an adenine deaminase-Cas9 fusion-induced deletion system (A-DEL) by combining Cas9 with rat APOBEC1 (rA1) and TadA 8e, respectively. Both C-DEL and A-DEL improve the efficiency of deletions compared with the conventional Cas9 system in human cells. In addition, the C-DEL system generates a considerable fraction of predictable multinucleotide deletions from 5'-deaminated C bases to the Cas9-cleavage site and increases the proportion of larger deletions at the target loci. Taken together, the C-DEL and A-DEL systems provide a practical strategy for producing efficient multinucleotide deletions, expanding the CRISPR/Cas9 toolsets for gene modifications in human cells.