Homology-based repair induced by CRISPR-Cas nucleases in mammalian embryo genome editing

Zhang, Xiya; Li, Tao; Ou, Jianping; Huang, Junjiu; Liang, Puping

doi:10.1007/s13238-021-00838-7

Cited by 25 publications

(20 citation statements)

References 149 publications

(287 reference statements)

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“…Cas9 specificity also relies on recognition of a short sequence (NGG) downstream of the gRNA, known as the protospacer adjacent motif (PAM), which is required for opening the DNA and target cleavage (Nishimasu et al., 2014; Sternberg, Redding, Jinek, Greene, & Doudna, 2014). After introduction of a DSB, two major DNA repair mechanisms can intervene: non‐homologous end joining (NHEJ) and homology‐directed repair (HDR) (Saleh‐Gohari & Helleday, 2004; Urnov, Rebar, Holmes, Zhang, & Gregory, 2010; Zhang, Li, Ou, Huang, & Liang, 2021). The first knocks out the targeted gene by generating nonspecific mutations (insertions/deletions, commonly known as indels) and tends to be error‐prone, while the second produces specific modifications determined by an exogenous donor template.…”

Section: Introductionmentioning

confidence: 99%

Efficient Cas9‐based Genome Editing Using CRISPR Analysis Webtools in Severe Early‐onset‐obesity Patient‐derived iPSCs

et al. 2022

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The CRISPR system is an adaptive defense mechanism used by bacteria and archaea against viruses and plasmids. The discovery of the CRISPR‐associated protein Cas9 and its RNA‐guided cleavage mechanism marked the beginning of a new era in genomic engineering by enabling the editing of a target region in the genome. Gene‐edited cells or mice can be used as models for understanding human diseases. Given its high impact in functional genomic experiments on different model systems, several CRISPR/Cas9 protocols have been generated in the past years. The technique uses a straightforward “cut and stitch” mechanism, but requires an accurate step‐by‐step design. One of the key points is the use of an efficient programmable guide RNA to increase the rate of success in obtaining gene‐specific edited clones. Here, we describe an efficient editing protocol using a ribonucleotide protein (RNP) complex for homology‐directed repair (HDR)–based correction of a point mutation in an induced pluripotent stem cell (iPSC) line generated from a 14‐year‐old patient with severe early‐onset obesity carrying a de novo variant of ARNT2. The resulting isogenic iPSC line, named CUIMCi003‐A‐1, has a normal karyotype, expresses stemness markers, and can be differentiated into progenies from all three germ layers. We provide a detailed workflow for designing a single guide RNA and donor DNA, and for isolating clonal human iPSCs edited with the desired modification. This article also focuses on parameters to consider when selecting reagents for CRISPR/Cas9 gene editing after testing their efficiency with in silico tools. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Design of sgRNAs and PCR primers Basic Protocol 2: Testing the efficiency of sgRNAs Basic Protocol 3: Design of template or donor DNA Basic Protocol 4: Targeted gene editing Basic Protocol 5: Selection of positive clones Basic Protocol 6: Freezing, thawing, and expansion of cells Basic Protocol 7: Characterization of edited cell lines

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Section: Introductionmentioning

confidence: 99%

Efficient Cas9‐based Genome Editing Using CRISPR Analysis Webtools in Severe Early‐onset‐obesity Patient‐derived iPSCs

et al. 2022

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“…In this strategy, the use of dsDNA as donor DNA produces false-positive cell clones via direct transcription and translation or via random integration into the genome through canonical NHEJ (c-NHEJ) [ 15 ]. Recent studies have demonstrated that ssDNA donors show superior performance compared to dsDNA donors in mammalian systems by reducing the probability of NHEJ [ 16 ].…”

Section: Resultsmentioning

confidence: 99%

Efficient gene editing through an intronic selection marker in cells

Wang

et al. 2022

Cell. Mol. Life Sci.

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Background Gene editing technology has provided researchers with the ability to modify genome sequences in almost all eukaryotes. Gene-edited cell lines are being used with increasing frequency in both bench research and targeted therapy. However, despite the great importance and universality of gene editing, the efficiency of homology-directed DNA repair (HDR) is too low, and base editors (BEs) cannot accomplish desired indel editing tasks. Results and discussion Our group has improved HDR gene editing technology to indicate DNA variation with an independent selection marker using an HDR strategy, which we named Gene Editing through an Intronic Selection marker (GEIS). GEIS uses a simple process to avoid nonhomologous end joining (NHEJ)-mediated false-positive effects and achieves a DsRed positive rate as high as 87.5% after two rounds of fluorescence-activated cell sorter (FACS) selection without disturbing endogenous gene splicing and expression. We re-examined the correlation of the conversion tract and efficiency, and our data suggest that GEIS has the potential to edit approximately 97% of gene editing targets in human and mouse cells. The results of further comprehensive analysis suggest that the strategy may be useful for introducing multiple DNA variations in cells.

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“…In this strategy, the use of dsDNA as donor DNA produces false-positive cell clones via direct transcription and translation or via random integration into the genome through canonical NHEJ (c-NHEJ) 14 . Recent studies have demonstrated that ssDNA donors show superior performance compared to dsDNA donors in mammalian systems by reducing the probability of NHEJ 15 .…”

Section: Resultsmentioning

confidence: 99%

Efficient Gene Editing Through an Intronic Selection Marker in Cells

Wang

et al. 2021

Preprint

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Background Gene editing technology has provided researchers with the ability to modify genome sequences in almost all eukaryotes. Gene-edited cell lines are being used with increasing frequency in both bench research and targeted therapy. Despite the great importance and universality of gene editing, however, precision and efficiency are hard to achieve with the prevailing editing strategies, such as homology-directed DNA repair (HDR) and the use of base editors (BEs). Results & Discussion Our group has developed a novel gene editing technology to indicate DNA variation with an independent selection marker using an HDR strategy, which we named Gene Editing through an Intronic Selection marker (GEIS). GEIS uses a simple process to avoid nonhomologous end joining (NHEJ)-mediated false-positive effects and achieves editing efficiency as high as 91% without disturbing endogenous gene splicing and expression. We re-examined the correlation of the conversion tract and editing efficiency, and our data suggest that GEIS has the potential to edit approximately 99% of gene editing targets in human and mouse cells. The results of further comprehensive analysis suggest that the strategy may be useful for introducing multiple DNA variations in cells.

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Homology-based repair induced by CRISPR-Cas nucleases in mammalian embryo genome editing

Cited by 25 publications

References 149 publications

Efficient Cas9‐based Genome Editing Using CRISPR Analysis Webtools in Severe Early‐onset‐obesity Patient‐derived iPSCs

Efficient Cas9‐based Genome Editing Using CRISPR Analysis Webtools in Severe Early‐onset‐obesity Patient‐derived iPSCs

Efficient gene editing through an intronic selection marker in cells

Efficient Gene Editing Through an Intronic Selection Marker in Cells

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