Adenine base editors induce off-target structure variations in mouse embryos and primary human T cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE280767 | GEO | 2024/11/01
REPOSITORIES: GEO
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Project description:CRISPR/Cas9-based genome editing has revolutionized experimental molecular biology and entered the clinical world for targeted gene therapy. Identifying DNA modifications occurring at CRISPR/Cas9 target sites is critical to determine efficiency and safety of editing tools. Here we show that insertions of LINE-1 (L1) retrotransposons can occur frequently at CRISPR/Cas9 editing sites. Together with PolyA-seq and an improved amplicon sequencing, we characterize more than 2,500 de novo L1 insertions at multiple CRISPR/Cas9 editing sites in HEK293T, HeLa and U2OS cells. These L1 retrotransposition events exploit CRISPR/Cas9-induced DSB formation and require L1 RT activity. Importantly, de novo L1 insertions are rare during genome editing by prime editors (PE), cytidine or adenine base editors (CBE or ABE), consistent with their reduced DSB formation. These data demonstrate that insertions of retrotransposons might be a potential outcome of CRISPR/Cas9 genome editing and provide further evidence on the safety of different CRISPR-based editing tools.
2022-06-07 | GSE178440 | GEO
Project description:CRISPR-guided DNA base editors enable the efficient installation of targeted single-nucleotide changes. Cytosine or adenine base editors (CBEs or ABEs), which are fusions of cytidine or adenosine deaminases to CRISPR-Cas nickases, can efficiently induce DNA C-to-T or A-to-G alterations in DNA, respectively. We recently demonstrated that both the widely used CBE BE3 (harboring a rat APOBEC1 cytidine deaminase) and the optimized ABEmax editor can induce tens of thousands of guide RNA-independent, transcriptome-wide RNA base edits in human cells with high efficiencies. In addition, we showed the feasibility of creating SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that exhibit substantially reduced unwanted RNA editing activities while retaining robust and more precise on-target DNA editing. Here we describe structure-guided engineering of SECURE-ABE variants that not only possess reduced off-target RNA editing with comparable on-target DNA activities but are also the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. In addition, we tested CBEs composed of cytidine deaminases other than APOBEC1 and found that human APOBEC3A (hA3A) cytidine deaminase CBE induces substantial transcriptome-wide RNA base edits with high efficiencies. By contrast, a previously described “enhanced” A3A (eA3A) cytidine deaminase CBE or a human activation-induced cytidine deaminase (hAID) CBE induce substantially reduced or near background levels of RNA edits. In sum, our work describes broadly useful SECURE-ABE and -CBE base editors and reinforces the importance of minimizing RNA editing activities of DNA base editors for research and therapeutic applications.
2019-07-11 | GSE129893 | GEO
Project description:CRISPR-guided DNA base editors enable the efficient installation of targeted single-nucleotide changes. Cytosine or adenine base editors (CBEs or ABEs), which are fusions of cytidine or adenosine deaminases to CRISPR-Cas nickases, can efficiently induce DNA C-to-T or A-to-G alterations in DNA, respectively. We recently demonstrated that both the widely used CBE BE3 (harboring a rat APOBEC1 cytidine deaminase) and the optimized ABEmax editor can induce tens of thousands of guide RNA-independent, transcriptome-wide RNA base edits in human cells with high efficiencies. In addition, we showed the feasibility of creating SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that exhibit substantially reduced unwanted RNA editing activities while retaining robust and more precise on-target DNA editing. Here we describe structure-guided engineering of SECURE-ABE variants that not only possess reduced off-target RNA editing with comparable on-target DNA activities but are also the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. In addition, we tested CBEs composed of cytidine deaminases other than APOBEC1 and found that human APOBEC3A (hA3A) cytidine deaminase CBE induces substantial transcriptome-wide RNA base edits with high efficiencies. By contrast, a previously described “enhanced” A3A (eA3A) cytidine deaminase CBE or a human activation-induced cytidine deaminase (hAID) CBE induce substantially reduced or near background levels of RNA edits. In sum, our work describes broadly useful SECURE-ABE and -CBE base editors and reinforces the importance of minimizing RNA editing activities of DNA base editors for research and therapeutic applications.
2019-07-11 | GSE129889 | GEO
Project description:CRISPR/Cas9 has been adapted to disrupt endogenous genes in adoptive T-lymphocyte therapy to prevent graft-versus-host disease. However, genome editing also generates prevalent deleterious structural variations (SVs), including chromosomal translocations and large deletions, raising safety concerns about reinfused T cells. Here, we dynamically monitored the progression of SVs in a mouse model of T-cell receptor (TCR)-transgenic T-cell adoptive transfer, mimicking TCR T therapeutics. Remarkably, CRISPR/Cas9-induced SVs persist and undergo clonal expansion in vivo after three weeks or even two months, evidenced by high enrichment and low junctional diversity of identified SVs post infusion. Specifically, we detected 128 expanded translocations, with 20,615 as the highest number of amplicons. The identified SVs are stochastically selected among different individuals and show an inconspicuous locus preference. Moreover, viral DNA integrations are routinely detected in edited T cells and also undergo clonal expansion as SVs do. The persistent SVs and viral DNA integrations in the infused T cells may constantly threaten genome integrity, drawing immediate attention to the safety of CRISPR/Cas9-engineered T cells mediated immunotherapy.
2022-10-12 | GSE202887 | GEO
Project description:CRISPR-Cas base editors are preferred tool for genome editing as they generate desired editing without any double strand break in the genome, as double stand break is detrimental to the cells. In our study we have demonstrated the significance of base editors in editing the highly homologous HBG promoter (HBG1 and HBG2) region to introduce novel HPFH-like mutation to elevate HbF for therapeutical applications. Previous studies revealed that the base editors can cause unintended Cas-independent edits at transcriptome level. To validate off-target at RNA level, we performed a transcriptome wide analysis. The frequency of unintended edits in the HUDEP-2 stable cell lines expressing the base editors with the gRNA were not significant compared to the control. We determined the ABE mediated A to I conversion and CBE mediated C to U conversion across the base edited samples. The RNA off-target analysis was carried out with the help of REDItools v 2 tool. This data suggests that despite high on-target editing in DNA, the Cas-independent RNA off-target were not at detectable range compared to control. The differential expression of 34 selected genes which necessitate globin regulation were compared between the unedited HUDEP WT, CBE control, ABE control, and edited ABE (with gRNA 2/11) and edited CBE (with gRNA 2/11). We observed that there is no significant differential gene expression between the edited and control cells except the gamma and delta globin genes. These results suggest that base editors are preferred tools to edit highly homologous HBG promoter region to created HPFH-like mutations inducing HbF levels without causing double strand breaks, larger deletions and no significant RNA off-targets which are detrimental to the gene edited cells.
2022-01-02 | GSE192801 | GEO
Project description:Plant biotechnology needs new methods that accelerate design-build-test-learn cycles to develop new gene editing reagents. We have established ITER (Iterative Testing of Editing Reagents) based on arrayed protoplast transfections and high-content imaging, allowing one optimization cycle –from design to results– within three weeks. We validated ITER in wheat and maize protoplasts using Cas9 cytosine and adenine base editors and used it to develop an optimized LbCas12a-ABE system. Sequential improvement of five components –NLS, crRNA, LbCas12a nuclease, adenine deaminase and linker– led to a systematic, stepwise increase in ABE activity at extrachromosomal GFP reporter (from 0.5% to 40%) and endogenous target sites. We confirmed the activity of LbCas12a-ABE in stable wheat transformants and leveraged these improvements to develop a highly mutagenic LbCas12a nuclease and a LbCas12a-CBE. Our data show that ITER is a sensitive, versatile, and high-throughput platform that can be harnessed to accelerate the development of genome editing technologies in plants.
2022-11-21 | GSE200450 | GEO
Project description:The Detect-seq provides an unbiased method for genome-wide identification of CBE induced off-targets inside of cells. Purpose: Cytosine base editors (CBEs) have the potential to correct human pathogenic point mutations. However, its genome-wide specificity remains poorly understood, precluding its therapeutic applications. Unbiased tools are urgently needed to comprehensively evaluate CBE off-targets at the genome-wide level. Methods: The genomic DNA from CBE transfected cells are processed with fragmentation, endogenous d5fC protection, damage repair, biotin-dUTP and 5fdCTP labeling, and pull-down steps. After the sequencing library preparation, the FASTQ files are produced by the Illumina HiSeq XTen platform. To guarantee the reproducibility, each case of the experiment is present with two biological replicates. Results: Through Detect-seq, we comprehensively profiled the genome-wide off-targets of CBE for several representative sgRNAs, and observed both weak, “random-like” off-targets as well as strong Cas9-dependent off-targets in different human cell lines. Cas9-dependent off-targets can be prevalent for several sgRNAs, and demonstrate a divergent degrees of sequence similarity to the sgRNA. These CBE-induced off-target sites differ greatly from those caused by Cas9 nuclease alone, suggesting inherently different properties among these genome editing tools. Targeted amplicon sequencing further corroborated the novel off-target sites by Detect-seq; quite a few sites showed editing ratio that is on the same order of magnitude or even comparable to the on-target sites. Detect-seq also revealed two unexpected types of Cas9-dependent off-targets, which are out-of-protospacer editing and target-strand editing. These novel off-targets were likely caused by an unstable structure at the PAM-distal side of the Cas9-sgRNA-DNA complex. Based upon such understanding to the off-target editing, we engineered several CBE variants bearing mutations in APOBEC1 and obtained improved CBEs with significantly reduced off-target editing activities.
2021-04-08 | GSE152907 | GEO
Project description:The Detect-seq provides an unbiased method for genome-wide identification of CBE induced off-targets inside of cells. Purpose: Cytosine base editors (CBEs) have the potential to correct human pathogenic point mutations. However, its genome-wide specificity remains poorly understood, precluding its therapeutic applications. Unbiased tools are urgently needed to comprehensively evaluate CBE off-targets at the genome-wide level. Methods: The genomic DNA from CBE transfected cells are processed with fragmentation, endogenous d5fC protection, damage repair, biotin-dUTP and 5fdCTP labeling, and pull-down steps. After the sequencing library preparation, the FASTQ files are produced by the Illumina HiSeq XTen platform. To guarantee the reproducibility, each case of the experiment is present with two biological replicates. Results: Through Detect-seq, we comprehensively profiled the genome-wide off-targets of CBE for several representative sgRNAs, and observed both weak, “random-like” off-targets as well as strong Cas9-dependent off-targets in different human cell lines. Cas9-dependent off-targets can be prevalent for several sgRNAs, and demonstrate a divergent degrees of sequence similarity to the sgRNA. These CBE-induced off-target sites differ greatly from those caused by Cas9 nuclease alone, suggesting inherently different properties among these genome editing tools. Targeted amplicon sequencing further corroborated the novel off-target sites by Detect-seq; quite a few sites showed editing ratio that is on the same order of magnitude or even comparable to the on-target sites. Detect-seq also revealed two unexpected types of Cas9-dependent off-targets, which are out-of-protospacer editing and target-strand editing. These novel off-targets were likely caused by an unstable structure at the PAM-distal side of the Cas9-sgRNA-DNA complex. Based upon such understanding to the off-target editing, we engineered several CBE variants bearing mutations in APOBEC1 and obtained improved CBEs with significantly reduced off-target editing activities. Detect-seq utilized a dU-mapping strategy to profile on-target and off-target editing events by CBE at the whole-genome level. We applied Detect-seq to off-target evaluation in HEK293T and MCF7 cells for BE4max with several frequently used sgRNAs: “VEGFA site 2”, “HEK293 site 4”, “EMX1”, and “RNF2”.
2021-04-08 | GSE151265 | GEO
Project description:The gain-of-function mutation of c.G6055A (p.G2019S) in Leucine-rich repeat kinase 2 (LRRK2) gene is the most prevalent genetic cause of Parkinson’s disease (PD). Although clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing has been used to generate isogenic control lines, there are risks of creating indels and genomic instability together with a lower efficiency in non-dividing cells. The recent advance of adenine base editors (ABEs) could convert targeted A•T base pairs to G•C base pairs without double-strand DNA breaks or donor DNA templates and function in post-mitotic cells. Here, we demonstrate a complete correction of an induced pluripotent stem cell (iPSC) line derived from a PD patient having LRRK2 p.G2019S mutation using variable genome editing methods, including CRISPR/Cas9-based homology-directed repair (HDR) and ABEs. The corrected isogenic iPSCs derived dopaminergic neurons showed a down-regulated LRRK2 kinase activity, decreased phospho-a-synuclein accumulations, reduced apoptosis and restored neurite shrinkage phenotypes. The mutation correction efficacy, off-target and indels rates between CRISPR/Cas9-HDR and ABE were compared. Among the 47 clones generated by HDR, 3 clones (6.4%) were on-targeted corrected, while ABE showed a much higher correction rate (13 of 53 clones, 24.5%). Whole genome sequencing analysis revealed that there are 27 clones of HDR (57.4%) having deletions but none in clones of ABE, albeit ABE created 14 clones (26.4%) having off-target missense mutations. RNA sequencing and proteomic analysis of the mutant line identified 2220 differentially expressed genes compared with its isogenic control. Enrichment analysis demonstrated an over-representation of PD relevant pathways, including calcium ion dependent exocytosis, synaptic transports well as potential novel targets relevant to PD pathophysiology. These results envision that ABE could directly correct the pathogenic PD mutation in iPSCs for exploring the earliest events in PD pathophysiology and providing dopaminergic neurons for future cell therapies.
2022-04-24 | PXD025602 | JPOST Repository
Project description:Ensuring genome safety during gene editing is crucial for clinical translation of the high-efficient CRISPR-Cas9 toolbox. Therefore, the undesired events including chromosomal translocations, vector integrations, and large deletions arising during therapeutic gene editing remain to be adequately addressed or tackled in vivo. Here, we apply CRISPR-Cas9TX in comparison to CRISPR-Cas9 to target Vegfa for the treatment of age-related macular degeneration (AMD) disease in a mouse model. AAV delivery of both CRISPR-Cas9 and CRISPR-Cas9TX can efficiently inhibit laser-induced neovascularization. Importantly, Cas9TX almost eliminates chromosomal translocations that occur at a frequency of approximately 1% in Cas9-edited mouse retinal cells. Strikingly, the widely observed AAV integration at the target Vegfa site is also greatly dropped from nearly 50% of edited events to the background level during Cas9TX editing. Our findings reveal that chromosomal structural variations routinely occur during in vivo genome editing and highlight Cas9TX as a superior form of Cas9 for in vivo gene disruption.
2022-11-30 | GSE218818 | GEO