Multiplex gene targeting in the mouse embryo using a Cas9-Cpf1 hybrid guide RNA

Oh, Seak Hee; Lee, Hye-Jin; Ahn, Mi Kyoung; Jeon, Mi Yeon; Yoon, Ji Yeol; Jung, Young Jin; Kim, Gyeong-Nam; Baek, In-Jeoung; Kim, Inki; Kim, Kyung Mo; Sung, Young Hoon

doi:10.1016/j.bbrc.2020.12.072

Cited by 3 publications

(3 citation statements)

References 27 publications

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“…To determine the presence of indel mutations in pooled or clonal cells, genomic DNA samples were isolated and T7 endonuclease I (T7E1) assays were conducted as previously described (Oh et al 2021 ). Briefly, genomic regions encompassing the target site were PCR-amplified, heteroduplex DNAs were generated by heat denaturation and reannealing, and products were purified using a PCR cleanup kit from iNtRON Biotechnology, Inc (Seongnam, Korea).…”

Section: Methodsmentioning

confidence: 99%

Immortalization of primary marmoset skin fibroblasts by CRISPR-Cas9-mediated gene targeting

Jeong

Cho

Kwak

et al. 2022

Animal Cells and Systems

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Immortalized cell lines can be used for diverse in vitro experiments, providing invaluable data before conducting in vivo studies Callithrix jacchus , the common marmoset, is a non-human primate model utilized for studying various human diseases. However, only a few immortalized marmoset cell lines are currently available. In the present study, we reveal that CRISPR-Cas9-mediated targeting of the p53 gene or CDKN2A locus is an effective means for immortalizing primary marmoset skin fibroblasts. In addition to frameshift mutations that result in premature stop codons, in-frame mutations potentially destroying the DNA-binding motif of p53 are frequently detected in immortalized cells. Like Cdkn2a -deficient mouse cells, CDKN2A -deficient marmoset cells express wild-type p53 proteins normally respond to genotoxic stresses, including adriamycin and etoposide. Taken together, these findings indicate that Cas9- mediated gene targeting of the p53 gene or CDKN2A locus is an effective tool for establishing immortalized marmoset cell lines with defined genetic alterations.

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Section: Methodsmentioning

confidence: 99%

Immortalization of primary marmoset skin fibroblasts by CRISPR-Cas9-mediated gene targeting

Jeong

Cho

Kwak

et al. 2022

Animal Cells and Systems

Self Cite

View full text Add to dashboard Cite

show abstract

“…microinjected CRISPR-Cas9 and hybrid single guide RNA (sgRNA) to generate compound knockout mice deficient in the Il10ra (interleukin 10 receptor subunit alpha) and Dr3 (tumor necrosis factor receptor superfamily, member 25 [Tnfrsf25]) genes, targeting a homology of the IL10RA mutation causing refractory inflammatory bowel disease in pediatric patients. 54 The substitution mutation resulted in simultaneous Il10ra and Dr3 mutations; 37.5% of transferred edited embryos produced F0 offspring, and this demonstrated that multiplex gene targeting is achievable using hybrid sgRNA. Zhang et al.…”

Section: Introductionmentioning

confidence: 94%

“…Common techniques for editing rodent embryos include superovulation of female mice prior to timed mating, collection of embryos by oviductal flushing, insertion of targeted CRISPR-expression vectors into the one-cell or two-cell embryos by electroporation or microinjection, followed by embryo transfer into pseudopregnant surrogate females. 47 , 50 , 51 , 52 , 53 , 54 , 55 , 56 Resultant viable F0 and F1 offspring have allowed the physical effects of gene knockouts to be appreciated and determinations made on the contribution of individual genes. Mizuno et al.…”

Section: Introductionmentioning

confidence: 99%