Preparation and electroporation of Cas12a/Cpf1-guide RNA complexes for introducing large gene deletions in mouse embryonic stem cells

Kissling, Lucas; Monfort, Asun; Swarts, Daan C.; Wutz, Anton; Jínek, Martin

doi:10.1016/bs.mie.2018.10.028

Cited by 17 publications

(20 citation statements)

References 40 publications

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“…To assess the reproducibility of small volume zygote electroporation, we aimed at generating deletions in three genes, Ubn1 , Ubn2 , and Rmb12 . For these genes no mutations have been described in mice to date, and we have identified efficient Cas12a-crRNA combinations in mouse ESC electroporation experiments in a previous study (Kissling et al 2019). Although a large number of Cas12a-crRNA combinations showed excellent cleavage of their DNA targets in vitro, only a subset of Cas12a-crRNA RNPs yielded efficient deletions in ESCs.…”

Section: Resultsmentioning

confidence: 98%

“…Although a large number of Cas12a-crRNA combinations showed excellent cleavage of their DNA targets in vitro, only a subset of Cas12a-crRNA RNPs yielded efficient deletions in ESCs. This discrepancy was not trivially explained by sequence context or local chromatin accessibility as nucleases targeting opposite strands in close proximity showed substantial differences as evidenced by assessing NHEJ mutations (Kissling et al 2019). This suggests that combinations of the accessibility, sequence preference, and repair processes might influence the effective rates in cellular systems with which deletions can be obtained.…”

Section: Resultsmentioning

confidence: 99%

“…Production of the Cas12a-NLS-NLS-eGFP-NLS fusion protein (Cas12a nuclease), design of crRNAs, verification of DNA cleavage activity in vitro and PCR analysis of genomic deletions in Ubn1 , Ubn2 , and Rbm12 were previously described (Kissling et al 2019). crRNA sequences are listed (Suppl.…”

Section: Methodsmentioning

confidence: 99%

“…ESC lines were genotyped by PCR reactions using primer pairs spanning the deletion, and flanking the cleavage sites as previously published (Kissling et al 2019). A PCR master mix was prepared on ice for the number of samples tested.…”

Section: Methodsmentioning

confidence: 99%

“…While the molecular mechanisms of Cas12a nucleases have been extensively characterized (Dong et al 2016; Fonfara et al 2016; Zetsche et al 2015), in vivo applications of Cas12a remain less well established compared to in vivo applications of Cas9. In a previous study, we have provided a protocol for generating large deletions in mouse ESCs by electroporation of Cas12a RNP complexes (Kissling et al 2019). For this, two CRISPR-Cas nucleases are designed that cleave the genome at separated sites within a gene locus such that loss of the intervening sequence will result in a deletion.…”

Section: Introductionmentioning

confidence: 99%

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Introducing gene deletions by mouse zygote electroporation of Cas12a/Cpf1

et al. 2019

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CRISPR-associated (Cas) nucleases are established tools for engineering of animal genomes. These programmable RNA-guided nucleases have been introduced into zygotes using expression vectors, mRNA, or directly as ribonucleoprotein (RNP) complexes by different delivery methods. Whereas microinjection techniques are well established, more recently developed electroporation methods simplify RNP delivery but can provide less consistent efficiency. Previously, we have designed Cas12a-crRNA pairs to introduce large genomic deletions in the Ubn1, Ubn2, and Rbm12 genes in mouse embryonic stem cells (ESC). Here, we have optimized the conditions for electroporation of the same Cas12a RNP pairs into mouse zygotes. Using our protocol, large genomic deletions can be generated efficiently by electroporation of zygotes with or without an intact zona pellucida. Electroporation of as few as ten zygotes is sufficient to obtain a gene deletion in mice suggesting potential applicability of this method for species with limited availability of zygotes.Electronic supplementary materialThe online version of this article (10.1007/s11248-019-00168-9) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Introducing gene deletions by mouse zygote electroporation of Cas12a/Cpf1

et al. 2019

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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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Electroporation of AsCpf1/RNP at the Zygote Stage is an Efficient Genome Editing Method to Generate Knock-Out Mice Deficient in Leukemia Inhibitory Factor

Kim

Park

et al. 2019

Tissue Eng Regen Med

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Preparation and electroporation of Cas12a/Cpf1-guide RNA complexes for introducing large gene deletions in mouse embryonic stem cells

Cited by 17 publications

References 40 publications

Introducing gene deletions by mouse zygote electroporation of Cas12a/Cpf1

Introducing gene deletions by mouse zygote electroporation of Cas12a/Cpf1

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

Electroporation of AsCpf1/RNP at the Zygote Stage is an Efficient Genome Editing Method to Generate Knock-Out Mice Deficient in Leukemia Inhibitory Factor

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