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, Yeon Sun; Kim, Gyeong Ryeong; Park, Soo‐Jin; Yang, Seung Chel; Park, So Hee; Won, Ji; Shin, Ha Eun; Song, Haengseok; Kim, Hye Ryun

doi:10.1007/s13770-019-00225-8

Cited by 9 publications

(3 citation statements)

References 38 publications

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“…CRISPR-Cas12a has been shown to achieve gene knockout in mice and gene targeting in plants, bacteria, and mammalian cells [36][37][38][39][40] . Although CRISPR-Cas12a also accomplished single-base editing in mice using short (180 bases) single-stranded oligoDNA as a donor 41,42 , thus far, no study has reported the generation of transgene knock-in mice by CRISPR-Cas12a.…”

Section: Discussionmentioning

confidence: 99%

Development of an in vivo cleavable donor plasmid for targeted transgene integration by CRISPR-Cas9 and CRISPR-Cas12a

Ishibashi

Maki²,

Kitano³

et al. 2022

Sci Rep

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The CRISPR-Cas system is widely used for genome editing of cultured cells and organisms. The discovery of a new single RNA-guided endonuclease, CRISPR-Cas12a, in addition to the conventional CRISPR-Cas9 has broadened the number of editable target sites on the genome. Here, we developed an in vivo cleavable donor plasmid for precise targeted knock-in of external DNA by both Cas9 and Cas12a. This plasmid, named pCriMGET_9-12a (plasmid of synthetic CRISPR-coded RNA target sequence-equipped donor plasmid-mediated gene targeting via Cas9 and Cas12a), comprises the protospacer-adjacent motif sequences of Cas9 and Cas12a at the side of an off-target free synthetic CRISPR-coded RNA target sequence and a multiple cloning site for donor cassette insertion. pCriMGET_9-12a generates a linearized donor cassette in vivo by both CRISPR-Cas9 and CRISPR-Cas12a, which resulted in increased knock-in efficiency in culture cells. This method also achieved > 25% targeted knock-in of long external DNA (> 4 kb) in mice by both CRISPR-Cas9 and CRISPR-Cas12a. The pCriMGET_9-12a system expands the genomic target space for transgene knock-in and provides a versatile, low-cost, and high-performance CRISPR genome editing tool.

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Section: Discussionmentioning

confidence: 99%

Development of an in vivo cleavable donor plasmid for targeted transgene integration by CRISPR-Cas9 and CRISPR-Cas12a

Ishibashi

Maki²,

Kitano³

et al. 2022

Sci Rep

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“…Multiple studies in numerous species have used electroporation to deliver CRISPR Cas9 genome-editing reagents into zygotes to generate knockout embryos and animals. Non-mosaic knockouts have been most efficiently produced in rats and mice (Hashimoto et al, 2016;Chen et al, 2019) targeting a wide range of genes, including LIF (Kim et al, 2020), Rad51 (Iwata et al, 2019), and Rosa26 (Troder et al, 2018).…”

Section: Electroporation-mediated Knockoutsmentioning

confidence: 99%

Electroporation-Mediated Genome Editing of Livestock Zygotes

Lin

Eenennaam

2021

Front. Genet.

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The introduction of genome editing reagents into mammalian zygotes has traditionally been accomplished by cytoplasmic or pronuclear microinjection. This time-consuming procedure requires expensive equipment and a high level of skill. Electroporation of zygotes offers a simplified and more streamlined approach to transfect mammalian zygotes. There are a number of studies examining the parameters used in electroporation of mouse and rat zygotes. Here, we review the electroporation conditions, timing, and success rates that have been reported for mice and rats, in addition to the few reports about livestock zygotes, specifically pigs and cattle. The introduction of editing reagents at, or soon after, fertilization can help reduce the rate of mosaicism, the presence of two of more genotypes in the cells of an individual; as can the introduction of nuclease proteins rather than mRNA encoding nucleases. Mosaicism is particularly problematic in large livestock species with long generation intervals as it can take years to obtain non-mosaic, homozygous offspring through breeding. Gene knockouts accomplished via the non-homologous end joining pathway have been more widely reported and successfully accomplished using electroporation than have gene knock-ins. Delivering large DNA plasmids into the zygote is hindered by the zona pellucida (ZP), and the majority of gene knock-ins accomplished by electroporation have been using short single stranded DNA (ssDNA) repair templates, typically less than 1 kb. The most promising approach to deliver larger donor repair templates of up to 4.9 kb along with genome editing reagents into zygotes, without using cytoplasmic injection, is to use recombinant adeno-associated viruses (rAAVs) in combination with electroporation. However, similar to other methods used to deliver clustered regularly interspaced palindromic repeat (CRISPR) genome-editing reagents, this approach is also associated with high levels of mosaicism. Recent developments complementing germline ablated individuals with edited germline-competent cells offer an approach to avoid mosaicism in the germline of genome edited founder lines. Even with electroporation-mediated delivery of genome editing reagents to mammalian zygotes, there remain additional chokepoints in the genome editing pipeline that currently hinder the scalable production of non-mosaic genome edited livestock.

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“…Three Cpf1 orthologs from Francisella novicida (FnCpf1), 28,[40][41][42] Acidaminococcus sp. (AsCpf1) 43,44 and Lachnospiraceae bacterium ND2006 (LbCpf1) [45][46][47] have been investigated intensively and are functional in mammalian cells (Table 1). As a type V system, Cpf1 recognizes a T-rich PAM, generates a staggered cut with a 5 0 -overhang and requires a single crRNA (no tracrRNA exists in natural CRISPR/Cpf1 systems).…”

mentioning

confidence: 99%

Get ready for the CRISPR/Cas system: A beginner's guide to the engineering and design of guide RNAs

Feng

et al. 2021

The Journal of Gene Medicine

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The clustered regularly interspaced short palindromic repeats (CRISPR) system is a state-of-the-art tool for versatile genome editing that has advanced basic research dramatically, with great potential for clinic applications. The system consists of two key molecules: a CRISPR-associated (Cas) effector nuclease and a single guide RNA.The simplicity of the system has enabled the development of a wide spectrum of derivative methods. Almost any laboratory can utilize these methods, although new users may initially be confused when faced with the potentially overwhelming abundance of choices. Cas nucleases and their engineering have been systematically reviewed previously. In the present review, we discuss single guide RNA engineering and design strategies that facilitate more efficient, more specific and safer gene editing.

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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

Cited by 9 publications

References 38 publications

Development of an in vivo cleavable donor plasmid for targeted transgene integration by CRISPR-Cas9 and CRISPR-Cas12a

Development of an in vivo cleavable donor plasmid for targeted transgene integration by CRISPR-Cas9 and CRISPR-Cas12a

Electroporation-Mediated Genome Editing of Livestock Zygotes

Get ready for the CRISPR/Cas system: A beginner's guide to the engineering and design of guide RNAs

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