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

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Abstract
CRISPR-Cas systems, including Cas9 and Cpf1 (Cas12a), are promising tools for generating gene knockout mouse models. Unlike Cas9, Cpf1 can generate multiple crRNAs from a single concatemeric crRNA precursor, which is favorable for multiplex gene editing. Recently, a hybrid guide RNA (hgRNA) system employing both Cas9 and Cpf1 was developed for multiplex gene editing. As the crRNA of Cpf1 was linked to the 3' end of the sgRNA for Cas9, it can be split into separate guide RNAs by Cpf1. To examine whether this Cas9-Cpf1 hybrid system is suitable for multiplex gene knockouts in the mouse embryo, we generated an hgRNA that simultaneously targets the mouse Il10ra gene by Cas9 and mouse Dr3 (or Tnfrsf25, death receptor3) gene by Cpf1. The expression of hgRNA from a single promoter induced significant indels at each gene in cultured mouse cells upon the co-expression of both Cas9 and Cpf1. Interestingly, the hgRNA exhibited comparable Cas9-mediated indel activity without Cpf1 expression. Similarly, when the hgRNA was co-microinjected with both Cas9 and Cpf1 mRNAs into mouse zygotes at the pronuclear stage, founder mice were generated harboring mutations in both the Il10ra and Dr3 genes. However, when Cas9 mRNA was used alone without Cpf1 mRNA, the mouse Il10ra gene targeting was significantly decreased. These results indicate that the hgRNA system is a possible tool for multiplex gene targeting in the mouse embryo.
Issued Date
2021
Type
Article
Keyword
Cas9Cpf1CRISPRHybrid guide RNAMultiplex gene targeting
DOI
10.1016/j.bbrc.2020.12.072
URI
https://oak.ulsan.ac.kr/handle/2021.oak/9558
https://ulsan-primo.hosted.exlibrisgroup.com/primo-explore/fulldisplay?docid=TN_cdi_proquest_miscellaneous_2476558683&context=PC&vid=ULSAN&lang=ko_KR&search_scope=default_scope&adaptor=primo_central_multiple_fe&tab=default_tab&query=any,contains,Multiplex%20gene%20targeting%20in%20the%20mouse%20embryo%20using%20a%20Cas9-Cpf1%20hybrid%20guide%20RNA&offset=0&pcAvailability=true
Publisher
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Location
미국
Language
한국어
ISSN
0006-291X
Citation Volume
539
Citation Start Page
48
Citation End Page
55
Appears in Collections:
Natural Science > Biological Sciences
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