Genome Editing Research Articles

Targeted Forward Genetics: Saturating Mutational Analyses of Specific Target Loci Within the Genome.

Precise allele replacement by homologous recombination (also known as "gene targeting" or "genome editing") allows scientists to engineer altered DNA sequences, insertions, or deletions at specific locations in the genome. Such reverse genetics provides powerful tools to elucidate the structure and function of regulatory DNA elements, genes, RNAs, and proteins within their natural, endogenous context. Here, we describe in detail the methodology for Targeted Forward Genetics (TFG), which supports population-scale, saturating screens of allele replacements spanning thousands of base pairs at a specific target locus in the genome. The overall approach and detailed protocols, developed for the fission yeast Schizosaccharomyces pombe, are extensible to other organisms in which gene targeting is feasible.

Fast Fission Yeast Genome Editing by CRISPR/Cas9 Using Gap Repair and Fluoride Selection.

We present a protocol to perform CRISPR/Cas9-mediated genome editing in the fission yeast Schizosaccharomyces pombe that does not require cloning and uses the fluoride exporter channel Fex1 as the selection marker. Transformation is typically carried out on the same day of PCR primer arrival and successfully edited strains are selected 5days after transformation. We expect the adoption of this protocol to further accelerate the throughput of genome editing in S. pombe.

CRISPR-Cas9 Genome Editing in Auxotrophic and Non-auxotrophic Fission Yeast Strains.

The CRISPR/Cas system is a very powerful genome-editing tool that has been developed over the past decade to optimize genome editing for many organisms. Here, we describe a rapid genome-editing method for fission yeast using the CRISPR-Cas9 system. It allows rapid generation of desired auxotrophic and non-auxotrophic strains without perturbing the local genome content by avoiding the insertion of selection markers at target loci.

Characterization and morphological development of oil palm transformed-callus on modified culture media

Genome editing through cisgenesis develops into scientific breakthroughs in accelerating oil palm breeding programs. However, one remaining problem is the low success of transformed-calli regeneration, while its scientific explanation is still underexplored. This study aimed to characterize and regenerate transformed-calli using various amino acids and antioxidants. Transformed callus that did not regenerate (un-regenerated transformed callus or UTC) after the transformation process was taken, then T-DNA integration was detected using the NPTII gene. Furthermore, the UTC was divided into four types based on morphological characteristics. The four types of UTCs were regenerated on media enriched with glutamine (for Type-1 callus), cysteine and putrescine (for Type-2 callus), and a combination of cysteine and ascorbic acid (for Type-3 and Type-4 callus). The research results obtained NPTII successfully amplified with a band size of 700bp. The results showed that on Type-1 callus, enrichment media with 10 mg L-1 L-glutamine could induce the formation of new nodular structures on UTC Type-1. On Type-2, media enriched with 5 mg L-1 L-cysteine + 20 mg L-1 putrescine increased the density of callus structures. Media enriched with 25 mg L-1 ascorbic acid + 25 mg L-1 L-cysteine could prevent the spread of brown callus on Type-3 callus, while Type-4 callus did not show any response and became dry. Our new findings will facilitate the basic research and unregenerated transformed callus and morphological callus development behavior in oil palm.

Multimodal scanning of genetic variants with base and prime editing.

Mutational scanning connects genetic variants to phenotype, enabling the interrogation of protein functions, interactions and variant pathogenicity. However, current methodologies cannot efficiently engineer customizable sets of diverse genetic variants in endogenous loci across cellular contexts in high throughput. Here, we combine cytosine and adenine base editors and a prime editor to assess the pathogenicity of a broad spectrum of variants in the epithelial growth factor receptor gene (EGFR). Using pooled base editing and prime editing guide RNA libraries, we install tens of thousands of variants spanning the full coding sequence of EGFR in multiple cell lines and assess the role of these variants in tumorigenesis and resistance to tyrosine kinase inhibitors. Our EGFR variant scan identifies important hits, supporting the robustness of the approach and revealing underappreciated routes to EGFR activation and drug response. We anticipate that multimodal precision mutational scanning can be applied broadly to characterize genetic variation in any genetic element of interest at high and single-nucleotide resolution.

Prime editing: A gene precision editing tool from inception to present.

Genetic mutations significantly contribute to the onset of diseases, with over half of the cases caused by single-nucleotide mutations. Advances in gene editing technologies have enabled precise editing and correction of mutated genes, offering effective treatment methods for genetic disorders. CRISPR/Cas9, despite its power, poses risks of inducing gene mutations due to DNA double-strand breaks (DSB). The advent of base editing (BE) and prime editing (PE) has mitigated these risks by eliminating the hazards associated with DNA DSBs, allowing for more precise gene editing. This breakthrough lays a solid foundation for the clinical application of gene editing technologies. This review discusses the principles, development, and applications of PE gene editing technology in various genetic mutation-induced diseases.

Amphiphilic Oligonucleotide Derivatives-Promising Tools for Therapeutics.

Recent advances in genetics and nucleic acid chemistry have created fundamentally new tools, both for practical applications in therapy and diagnostics and for fundamental genome editing tasks. Nucleic acid-based therapeutic agents offer a distinct advantage of selectively targeting the underlying cause of the disease. Nevertheless, despite the success achieved thus far, there remain unresolved issues regarding the improvement of the pharmacokinetic properties of therapeutic nucleic acids while preserving their biological activity. In order to address these challenges, there is a growing focus on the study of safe and effective delivery methods utilising modified nucleic acid analogues and their lipid bioconjugates. The present review article provides an overview of the current state of the art in the use of chemically modified nucleic acid derivatives for therapeutic applications, with a particular focus on oligonucleotides conjugated to lipid moieties. A systematic analysis has been conducted to investigate the ability of amphiphilic oligonucleotides to self-assemble into micelle-like structures, as well as the influence of non-covalent interactions of such derivatives with serum albumin on their biodistribution and therapeutic effects.

CRISPR-AsCas12f1 couples out-of-protospacer DNA unwinding with exonuclease activity in the sequential target cleavage.

Type V-F CRISPR-Cas12f is a group of hypercompact RNA-guided nucleases that present a versatile in vivo delivery platform for gene therapy. Upon target recognition, Acidibacillus sulfuroxidans Cas12f (AsCas12f1) distinctively engenders three DNA break sites, two of which are located outside the protospacer. Combining ensemble and single-molecule approaches, we elucidate the molecular details underlying AsCas12f1-mediated DNA cleavages. We find that following the protospacer DNA unwinding and non-target strand (NTS) DNA nicking, AsCas12f1 surprisingly carries out bidirectional exonucleolytic cleavage from the nick. Subsequently, DNA unwinding is extended to the out-of-protospacer region, and AsCas12f1 gradually digests the unwound DNA beyond the protospacer. Eventually, the single endonucleolytic target-strand DNA cleavage at 3 nt downstream of the protospacer readily dissociates the ternary AsCas12f1-sgRNA-DNA complex from the protospacer adjacent motif-distal end, leaving a staggered double-strand DNA break. The coupling between the unwinding and cleavage of both protospacer and out-of-protospacer DNA is promoted by Mg2+. Kinetic analysis on the engineered AsCas12f1-v5.1 variant identifies the only accelerated step of the protospacer NTS DNA trimming within the sequential DNA cleavage. Our findings provide a dynamic view of AsCas12f1 catalyzing DNA unwinding-coupled nucleolytic cleavage and help with practical improvements of Cas12f-based genome editing tools.

Advances in machine learning methods in copper alloys: a review.

Advanced copper and copper alloys, as significant engineering structural materials, have recently been extensively used in energy, electron, transportation, and aviation domains. Higher requirements urge the emergence of high-performance copper alloys. However, the traditional trial-and-error experimental observations and computational simulation research used to design and develop novel materials are time-consuming and costly. With the accumulation of material research and rapid development of computational ability, the thorough application of material genome engineering has sped up the development of novel materials and facilitates the process of systematic engineering application. This review summarizes the benefits of data-driven machine learning techniques and the state of the art of machine learning research in the area of copper alloys. It also displays the widely used computational simulation approaches (e.g., the first-principles calculation, molecular dynamics simulation, phase-field simulations, and finite element analysis) and their combined applications in material design and property prediction. Finally, the limitations of machine learning research methods are outlined, and future development directions are proposed.

ReaL-MGE is a tool for enhanced multiplex genome engineering and application to malonyl-CoA anabolism

The complexities encountered in microbial metabolic engineering continue to elude prediction and design. Unravelling these complexities requires strategies that go beyond conventional genetics. Using multiplex mutagenesis with double stranded (ds) DNA, we extend the multiplex repertoire previously pioneered using single strand (ss) oligonucleotides. We present ReaL-MGE (Recombineering and Linear CRISPR/Cas9 assisted Multiplex Genome Engineering). ReaL-MGE enables precise manipulation of numerous large DNA sequences as demonstrated by the simultaneous insertion of multiple kilobase-scale sequences into E. coli, Schlegelella brevitalea and Pseudomonas putida genomes without any off-target errors. ReaL-MGE applications to enhance intracellular malonyl-CoA levels in these three genomes achieved 26-, 20-, and 13.5-fold elevations respectively, thereby promoting target polyketide yields by more than an order of magnitude. In a further round of ReaL-MGE, we adapt S. brevitalea to malonyl-CoA elevation utilizing a restricted carbon source (lignocellulose from straw) to realize production of the anti-cancer secondary metabolite, epothilone from lignocellulose. Multiplex mutagenesis with dsDNA enables the incorporation of lengthy segments that can fully encode additional functions. Additionally, the utilization of PCR to generate the dsDNAs brings flexible design advantages. ReaL-MGE presents strategic options in microbial metabolic engineering.

Enhancing genomic disorder prediction through Feynman Concordance and Interpolated Nearest Centroid techniques

Clinical biomedical applications of genomic technologies are extensive and provide possibilities to enhance healthcare covering the span of medical talents. Genome disorder prediction is an important issue in biomedical research. Genome disorders cause multivariate diseases such as cancer, dementia, diabetes, Leigh syndrome, etc. Existing machine and deep learning-based methods were introduced to forecast genome disorders. However, the genome prediction outcomes were not sufficient. To address this issue, propose a new method called Quadratic Feynman Polynomial Interpolated and Vector Nearest Centroid-based (QFPI-VNC) for acutely predicting the genome disorder with improved sensitivity and specificity. First, we utilized medical data about children from a public genomes dataset and applied it to Linear Quadratic and Feynman Kac Genome filtering to obtain computationally efficient filtered results. Next, the results are fed to the Concordance Correlated Polynomial Interpolation with the purpose of extracting genome wide data in an accurate manner. Finally, the features extracted are fused and fed to the Support Vector and Nearest Centroid model for genome disorder prediction. Experimental investigations of the proposed method employing the genome dataset confirm that the performance of the proposed method is prospective and in the scope of acceptance with relative to state-of-the-art methods in terms of convergence speed, recognition rate, sensitivity, and specificity. Results suggest that the QFPI-VNC method produces the best performance with a higher genome disease detection rate by 14%, accuracy by 11%, sensitivity by 14% specificity by 12%, and lesser convergence speed by 29% than compared to state-of-the-art methods.

PolyASite v3.0: a multi-species atlas of polyadenylation sites inferred from single-cell RNA-sequencing data.

The broadly used 10X Genomics technology for single-cell RNA sequencing (scRNA-seq) captures RNA 3' ends. Thus, some reads contain part of the non-templated polyadenosine tails, providing direct evidence for the sites of 3' end cleavage and polyadenylation on the respective RNAs. Taking advantage of this property, we recently developed the SCINPAS workflow to infer polyadenylation sites (PASs) from scRNA-seq data. Here, we used this workflow to construct version 3.0 (v3.0, https://polyasite.unibas.ch/) of the PolyASite Atlas from a big compendium of publicly available human, mouseand worm scRNA-seq datasets obtained from healthy tissues. As the resolution of scRNA-seq was too low for robust detection of cell-level differences in PAS usage, we aggregated samples based on their tissue-of-origin to construct tissue-level catalogs of PASs. These provide qualitatively new information about PAS usage, in comparison to the previous PAS catalogs that were based on bulk 3' end sequencing experiments primarily in cell lines. In the new version, we document stringency levels associated with each PAS so that users can balance sensitivity and specificity in their analysis. We also upgraded the integration with the UCSC Genome Browser and developed track hubs conveniently displaying pooled and tissue-specific expression of PASs.

Efficient DNA-free co-targeting of nuclear genes in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii, a model organism for unicellular green microalgae, is widely used in basic and applied research. Nonetheless, proceeding towards synthetic biology requires a full set of manipulation techniques for inserting, removing, or editing genes. Despite recent advancements in CRISPR/Cas9, still significant limitations in producing gene knock-outs are standing, including (i) unsatisfactory genome editing (GE) efficiency and (ii) uncontrolled DNA random insertion of antibiotic resistance markers. Thus, obtaining efficient gene targeting without using marker genes is instrumental in developing a pipeline for efficient engineering of strains for biotechnological applications. We developed an efficient DNA-free gene disruption strategy, relying on phenotypical identification of mutants, to (i) precisely determine its efficiency compared to marker-relying approaches and (ii) establish a new DNA-free editing tool. This study found that classical CRISPR Cas9-based GE for gene disruption in Chlamydomonas reinhardtii is mainly limited by DNA integration. With respect to previous results achieved on synchronized cell populations, we succeeded in increasing the GE efficiency of single gene targeting by about 200 times and up to 270 times by applying phosphate starvation. Moreover, we determined the efficiency of multiplex simultaneous gene disruption by using an additional gene target whose knock-out did not lead to a visible phenotype, achieving a co-targeting efficiency of 22%. These results expand the toolset of GE techniques and, additionally, lead the way to future strategies to generate complex genotypes or to functionally investigate gene families. Furthermore, the approach provides new perspectives on how GE can be applied to (non-) model microalgae species, targeting groups of candidate genes of high interest for basic research and biotechnological applications.

Artificial Insemination as a Possible Convenient Tool to Acquire Genome-Edited Mice via In Vivo Fertilization with Engineered Sperm.

Advances in genome editing technology have made it possible to create genome-edited (GE) animals, which are useful for identifying isolated genes and producing models of human diseases within a short period of time. The production of GE animals mainly relies on the gene manipulation of pre-implantation embryos, such as fertilized eggs and two-cell embryos, which can usually be achieved by the microinjection of nucleic acids, electroporation in the presence of nucleic acids, or infection with viral vectors, such as adeno-associated viruses. In contrast, GE animals can theoretically be generated by fertilizing ovulated oocytes with GE sperm. However, there are only a few reports showing the successful production of GE animals using GE sperm. Artificial insemination (AI) is an assisted reproduction technology based on the introduction of isolated sperm into the female reproductive tract, such as the uterine horn or oviductal lumen, for the in vivo fertilization of ovulated oocytes. This approach is simpler than the in vitro fertilization-based production of offspring, as the latter always requires an egg transfer to recipient females, which is labor-intensive and time-consuming. In this review, we summarize the various methods for AI reported so far, the history of sperm-mediated gene transfer, a technology to produce genetically engineered animals through in vivo fertilization with sperm carrying exogenous DNA, and finally describe the possibility of AI-mediated creation of GE animals using GE sperm.

In vivo Treatment of a Severe Vascular Disease via a Bespoke CRISPR-Cas9 Base Editor.

Genetic vascular disorders are prevalent diseases that have diverse etiologies and few treatment options. Pathogenic missense mutations in the alpha actin isotype 2 gene ( ACTA2 ) primarily affect smooth muscle cell (SMC) function and cause multisystemic smooth muscle dysfunction syndrome (MSMDS), a genetic vasculopathy that is associated with stroke, aortic dissection, and death in childhood. Here, we explored genome editing to correct the most common MSMDS-causative mutation ACTA2 R179H. In a first-in-kind approach, we performed mutation-specific protein engineering to develop a bespoke CRISPR-Cas9 enzyme with enhanced on-target activity against the R179H sequence. To directly correct the R179H mutation, we screened dozens of configurations of base editors (comprised of Cas9 enzymes, deaminases, and gRNAs) to develop a highly precise corrective A-to-G edit with minimal deleterious bystander editing that is otherwise prevalent when using wild-type SpCas9 base editors. We then created a murine model of MSMDS that exhibits phenotypes consistent with human patients, including vasculopathy and premature death, to explore the in vivo therapeutic potential of this base editing strategy. Delivery of the customized base editor via an engineered SMC-tropic adeno-associated virus (AAV-PR) vector substantially prolonged survival and rescued systemic phenotypes across the lifespan of MSMDS mice, including in the vasculature, aorta, and brain. Together, our optimization of a customized base editor highlights how bespoke CRISPR-Cas enzymes can enhance on-target correction while minimizing bystander edits, culminating in a precise editing approach that may enable a long-lasting treatment for patients with MSMDS.

DDDR-09. POTENTIAL OF GSPT1 AS A NOVEL TARGET FOR GLIOBLASTOMA THERAPY

Abstract Glioblastoma is the most common malignant brain tumor in adults, the survival rate of which has not significantly improved over the past three decades. Therefore, there is an urgent need to develop novel treatment modalities. We have reported G1 to S phase transition 1 (GSPT1) as a novel downstream target of RAC1, which is a tumor driving and promoting molecule, and that depletions of GSPT1 as well as RAC1 induced delayed cell cycle progression in primary astrocytes. Herein, we examined the potential of GSPT1 as a novel target for glioblastoma therapy. CC-885, a cereblon modulator that degrades GSPT1 by bridging GSPT1 to CRL4 E3 ubiquitin ligase complex, was administered to nude mice with transplanted brain tumors of U87 glioblastoma cells. The survival period was significantly longer in CC-885 treated mice than in control mice. Furthermore, we generated GSPT1-knockout (KO) U87 cells by genome editing and GSPT1-KO U87 cells with stable overexpression of FLAG-tagged GSPT1 (Rescued GSPT1-KO U87 cells). Mice with transplanted GSPT1-KO U87 cells and Rescued GSPT1-KO U87 cells showed significantly longer and similar survival periods, respectively, as those with WT U87 cells. GSPT1-KO U87 cells showed enhanced apoptosis, detected by cleaved PARP1, compared to WT U87 cells. Brain tumors due to transplantation of GSPT1-KO U87 cells also showed enhanced apoptosis compared to those due to transplantation of WT and Rescued GSPT1-KO U87 cells. GSPT1 expression was confirmed in patients with glioblastoma. However, the clinical study using 87 glioblastoma samples showed that GSPT1 mRNA levels were not associated with overall survival. Taken together, we propose that GSPT1 is an essential protein for glioblastoma growth, but not its malignant characteristics, and that GSPT1 is a potential target for developing glioblastoma therapeutics.

CSIG-28. DISSECTING THE FUNCTIONAL IMPAIRMENT OF WILD-TYPE P53 IN HUMAN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most common malignant brain tumor in adults and is characterized by heterogeneous nature, invasive potential, and poor treatment response. Work from The Cancer Genome Atlas (TCGA) identified the tumor suppressor gene TP53 as a GBM driver. In contrast to several cancers, only ~35% of GBM cases carry mutations in TP53. Work from our lab showed that TP53 mutations emerge as ‘late’ events during GBM growth. Therefore, we hypothesized that wild-type (wt) p53 function is impaired in GBM. We used the TCGA data of 591 GBM patients to compare the clinical and molecular characteristics of wt and mutant TP53 patients. Gene expression data were analyzed to compare the correlation of TP53 with the expression of key p53 regulators and identify differentially expressed regulators between our groups. Furthermore, we evaluated the expression of p53 and selected regulators in patient-derived cells (PDCs) and control lines with known TP53 status. Our results show that wt TP53 patients live less than mutant patients and both groups respond similarly to treatment. No differences in age, sex, race, TP53 levels, and MGMT methylation status were observed between the groups. Gene expression analyses revealed that MAPK8, a p53 activator, negatively correlates with TP53 in the wt group. Moreover, ~21% of p53 regulators are differentially expressed between wt and mutant patients. Among these, we selected SIRT3, a p53 repressor, for experimental validation. We verified the p53 genetic status of PDCs through western blot and confirmed Sirt3 expression in wt and mutant TP53 PDCs. By CRISPR-Cas9-based genomic editing, we are currently evaluating the impact of SIRT3 knockout in GBM-PDC and patient-derived xenografts. We expect that SIRT3 knockout will restore p53 function in the wt group. In conclusion, our results suggest that p53 function is impaired in wt patients and provide insights into mechanisms affecting its activity.

Breeding and biotechnology approaches to enhance the nutritional quality of rapeseed byproducts for sustainable alternative protein sources- a critical review.

Global protein consumption is increasing exponentially, which requires efficient identification of potential, healthy, and simple protein sources to fulfil the demands. The existing sources of animal proteins are high in fat and low in fiber composition, which might cause serious health risks when consumed regularly. Moreover, protein production from animal sources can negatively affect the environment, as it often requires more energy and natural resources and contributes to greenhouse gas emissions. Thus, finding alternative plant-based protein sources becomes indispensable. Rapeseed is an important oilseed crop and the world's third leading oil source. Rapeseed byproducts, such as seed cakes or meals, are considered the best alternative protein source after soybean owing to their promising protein profile (30%-60% crude protein) to supplement dietary requirements. After oil extraction, these rapeseed byproducts can be utilized as food for human consumption and animal feed. However, anti-nutritional factors (ANFs) like glucosinolates, phytic acid, tannins, and sinapines make them unsuitable for direct consumption. Techniques like microbial fermentation, advanced breeding, and genome editing can improve protein quality, reduce ANFs in rapeseed byproducts, and facilitate their usage in the food and feed industry. This review summarizes these approaches and offers the best bio-nutrition breakthroughs to develop nutrient-rich rapeseed byproducts as plant-based protein sources.

TMOD-26. GENERATION OF A CHROMOSOME 8 GAIN;NF1-/- HIPSC-DERIVED SCHWANN CELL PRECURSOR MODEL

Abstract Neurofibromatosis type 1 (NF1) is an autosomal dominant cancer predisposition syndrome caused by a germline mutation in the NF1 gene. The loss of the second copy of NF1 within Schwann cell precursors (SCPs) contributes to the formation of plexiform neurofibromas (PNs) found in 30-50% of NF1 patients. PNs can progress into malignant peripheral sheath tumors (MPNSTs), an aggressive form of cancer and a leading cause of mortality in NF1 patients. Our lab previously demonstrated frequent chromosome 8 (Chr8) gain in NF1-MPNST patient-derived xenografts (PDX) and patient tumors. To investigate Chr8 gain’s role, we created isogenic NF1-/- iPSC cell lines with and without Chr8 gain. Trisomy 8 fibroblasts were obtained and single-cell cloned to establish wildtype (WT) and Chr8 gain populations. Washington University’s Genome Engineering & Stem Cell Center (GESC@MGI) reprogrammed these cells into human induced pluripotent stem cells (hiPSCs). Using synthetic gRNA and CRISPR/Cas9, NF1 knockout lines were generated. Four iPSC cell lines were differentiated into SCPs, and cell survival and proliferation were assessed using Incucyte cell survival and CellTiter-Glo® Assay. Fluorescence in situ hybridization (FISH) and karyotyping validated the human iPSCs. qPCR revealed high expression of iPSC markers OCT3/4 and SOX2 in all iPSC lines. After differentiation, SCP markers (GAP43 and ITGA4) were upregulated in WT, NF1-/-, and Chr8 gain;NF1-/- SCP lines, demonstrating successful differentiation. Furthermore, Chr8 gain; NF1-/- SCPs displayed enhanced proliferation and increased cell survival compared to WT and NF1-/- SCPs, suggesting improved characteristics in the presence of Chr8 gain. In summary, our research provides insights into Chr8 gain and NF1 loss effects on SCP differentiation, proliferation, and survival. Understanding these molecular mechanisms may contribute to developing targeted therapies for NF1.

Adenine base editors induce off-target structure variations in mouse embryos and primary human T cells

BackgroundThe safety of CRISPR-based gene editing methods is of the utmost priority in clinical applications. Previous studies have reported that Cas9 cleavage induced frequent aneuploidy in primary human T cells, but whether cleavage-mediated editing of base editors would generate off-target structure variations remains unknown. Here, we investigate the potential off-target structural variations associated with CRISPR/Cas9, ABE, and CBE editing in mouse embryos and primary human T cells by whole-genome sequencing and single-cell RNA-seq analyses.ResultsThe results show that both Cas9 and ABE generate off-target structural variations (SVs) in mouse embryos, while CBE induces rare SVs. In addition, off-target large deletions are detected in 32.74% of primary human T cells transfected with Cas9 and 9.17% of cells transfected with ABE. Moreover, Cas9-induced aneuploid cells activate the P53 and apoptosis pathways, whereas ABE-associated aneuploid cells significantly upregulate cell cycle-related genes and are arrested in the G0 phase. A percentage of 16.59% and 4.29% aneuploid cells are still observable at 3 weeks post transfection of Cas9 or ABE. These off-target phenomena in ABE are universal as observed in other cell types such as B cells and Huh7. Furthermore, the off-target SVs are significantly reduced in cells treated with high-fidelity ABE (ABE-V106W).ConclusionsThis study shows both CRISPR/Cas9 and ABE induce off-target SVs in mouse embryos and primary human T cells, raising an urgent need for the development of high-fidelity gene editing tools.