Multiplex gene targeting in the mouse embryo using a Cas9-Cpf1 hybrid guide RNA
Autor: | Young Hoon Sung, Hye Jin Lee, In-Jeoung Baek, Kyung Mo Kim, Mi Yeon Jeon, Mi Kyoung Ahn, Jeong-Soo Yoon, Seak Hee Oh, Inki Kim, Yeon Ju Jung, Gyeong-Nam Kim |
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Rok vydání: | 2021 |
Předmět: |
0301 basic medicine
Biophysics Biology Biochemistry Cell Line Mice 03 medical and health sciences 0302 clinical medicine CRISPR-Associated Protein 9 Animals Multiplex Guide RNA Molecular Biology Gene Gene knockout Gene Editing Trans-activating crRNA Mice Inbred ICR Messenger RNA Cas9 Gene targeting Cell Biology Embryo Mammalian Endonucleases Cell biology Mice Inbred C57BL 030104 developmental biology 030220 oncology & carcinogenesis Gene Targeting RNA Guide Kinetoplastida |
Zdroj: | Biochemical and Biophysical Research Communications. 539:48-55 |
ISSN: | 0006-291X |
Popis: | 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. |
Databáze: | OpenAIRE |
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