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On- and off-target effects of paired CRISPR-Cas nickase in primary human cells - long-read

ABSTRACT: Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in therapeutic genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double nickase-based strategies to generate staggered DNA double breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-Seq pipeline, D-CAST, to characterize chromosomal rearrangements induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from epidermolysis bullosa patients. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following single or paired Cas9-based nickase editing. Conversely, whereas single nickase applications did not result in gross genomic aberrations, the double nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects, but still leave substantial chromosomal rearrangements at on-target sites.

ORGANISM(S): Homo sapiens

PROVIDER: GSE241779 | GEO | 2024/03/15

REPOSITORIES: GEO

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On- and off-target effects of paired CRISPR-Cas nickase in primary human cells - long read

Project description:Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in therapeutic genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double nickase-based strategies to generate staggered DNA double breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-Seq pipeline, D-CAST, to characterize chromosomal rearrangements induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from epidermolysis bullosa patients. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following single or paired Cas9-based nickase editing. Conversely, whereas single nickase applications did not result in gross genomic aberrations, the double nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects, but still leave substantial chromosomal rearrangements at on-target sites.

2024-03-15 | GSE254922 | GEO

On- and off-target effects of paired CRISPR-Cas nickase in primary human cells - Amplicon sequencing

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On- and off-target effects of paired CRISPR-Cas nickase in primary human cells [Amplicon]

Project description:Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double nickase-based strategies to generate staggered DNA double-strand breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-Seq pipeline, D-CAST, to characterize chromosomal aberrations induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from epidermolysis bullosa patients. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following paired nickase editing. While the double nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases, the chromosomal on-target aberrations were qualitatively different and included a high proportion of insertions. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects, but still leave substantial chromosomal aberrations at on-target sites.

2024-03-15 | GSE255081 | GEO

Expanding the editable genome and CRISPR-Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking

Project description:Genome editing typically involves recombination between donor nucleic acids and genomic sequences subjected to double-stranded DNA breaks (DSBs) made by programmable nucleases (e.g. CRISPR-Cas9). Yet, amongst other deleterious by-products, DSBs yield translocations, off-target mutations and, most pervasively, unpredictable on-target allelic disruptions. Remarkably, hitherto, the untoward phenotypic consequences of on-target disruptions at allelic and non-allelic (e.g. pseudogene) sequences have received scant scrutiny and, crucially, remain to be addressed. Here, we demonstrate that gene-edited cells can lose fitness due to on-target DSBs and report that simultaneous single-stranded DNA break formation at donor and target DNA by CRISPR-Cas9 “nickases” overcomes, to a great extent, such genotype-phenotype disrupting events. Moreover, in trans paired nicking gene editing can efficiently and precisely add large DNA segments (i.e. live-cell reporter tags) into essential and multiple-copy genomic sequences while circumventing most of the allelic and non-allelic collateral mutations and chromosomal rearrangements characteristic of nuclease-dependent gene editing procedures.

2019-12-04 | GSE135064 | GEO

Quantitative evaluation of chromosomal rearrangements in primary gene-edited human stem cells by preclinical CAST-Seq

Project description:Genome editing with programmable nucleases has shown great promise for clinical translation but also revealed the risk of genotoxicity caused by chromosomal translocations or the insertion of mutations at off-target sites. Here, we describe CAST-Seq, an innovative assay to identify and quantify chromosomal aberrations derived from on- and off-target activities of CRISPR-Cas nucleases or TALENs. CAST-Seq also detected novel types of chromosomal rearrangements, including homology-mediated translocations that are mediated by homologous recombination. Depending on the employed designer nuclease, translocations occurred in 0–0.5% of gene-edited human stem cells and some 20% of target loci harbored gross aberrations. In conclusion, CAST-Seq analyses are particularly relevant for therapeutic editing of stem cells to enable a thorough risk assessment before clinical application of gene editing products.

2021-02-14 | GSE164389 | GEO

MiSeq paired-end sequencing of HEK293T cells stably expressing a Cas9/gRNA-dependent editable reporter vector to test novel nucleobase-editing techniques

Project description:The goal of these experiments were to test the on-target and target-adjacent editing efficiencies of different single-nucleobase editing systems. Previous studies have shown that tethering DNA mutating enzymes to Cas9-nickase-UGI complexes results in editing of chromosomal DNA. However, these editing events encompass undesirable target-adjacent nucleobase edits. Here, we characterize a novel approach that reduces the frequency of target-adjacent editing while maintaining a high level of on-target editing.

2020-02-23 | E-MTAB-8742 | biostudies-arrayexpress

Precise and broad scope genome editing based on high-specificity Cas9 nickases

Project description:RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off- target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by “tiptoeing” over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of genome editing procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.

2020-12-08 | GSE153471 | GEO

Comprehensive assessment of miniature CRISPR-Cas12f nucleases for gene disruption

Project description:Because of their smallness, the recently developed CRISPR-Cas12f nucleases can be effectively packaged into adeno-associated viruses for gene therapy. However, a systematic evaluation of the editing outcomes of CRISPR-Cas12f is lacking. In this study, we apply a high-throughput sequencing method to comprehensively assess the editing efficiency, specificity, and safety of four Cas12f proteins in parallel with that of Cas9 and two Cas12a proteins at multiple genomic sites. Cas12f nucleases achieve robust cleavage at most of the tested sites and mainly produce deletional fragments. In contrast, Cas9 and Cas12a show relatively higher editing efficiency at the vast majority of the tested sites. However, the off-target hotspots identified in the Cas9- and Cas12a-edited cells are negligibly detected in the Cas12f-edited cells. Moreover, compared to Cas9 and Cas12a nucleases, Cas12f nucleases reduce the levels of chromosomal translocations, large deletions, and integrated vectors by 2- to 3-fold. Therefore, our findings confirm the editing capacity of Cas12f and reveal the ability of this nuclease family to preserve genome integrity during genome editing.

2022-09-12 | GSE213149 | GEO

T-CAST: An optimized CAST-Seq pipeline for TALEN reveals superior safety and efficacy of obligate-heterodimeric scaffolds

Project description:Transcription activator-like effector nucleases (TALENs) are programmable nucleases that have entered the clinical stage. Each subunit of the dimer consists of a DNA-binding domain composed of an array of TALE repeats fused to the catalytically active portion of the FokI endonuclease. Upon DNA-binding of both TALEN arms in close proximity, the FokI domains dimerize and induce a staggered-end DNA double strand break. In this present study, we describe the implementation and validation of TALEN-specific CAST-Seq (T-CAST), a pipeline based on CAST-Seq that identifies TALEN-mediated off-target effects, nominates off-target sites with high fidelity, and predicts the TALEN pairing conformation leading to off-target cleavage. We validated T-CAST by assessing off-target effects of two promiscuous TALENs designed to target the CCR5 and TRAC loci. Expression of these TALENs caused high levels of translocations between the target sites and various off-target sites in primary T cells. Introduction of amino acid substitutions to the FokI domains, which render TALENs obligate-heterodimeric (OH-TALEN), mitigated the aforementioned off-target effects without loss of on-target activity. Our findings highlight the significance of T-CAST to assess off-target effects of TALEN designer nucleases and to evaluate mitigation strategies, and advocate the use of obligate-heterodimeric TALEN scaffolds for therapeutic genome editing.

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