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

Multiplex gene targeting in the mouse embryo using a Cas9-Cpf1 hybrid guide RNA. Biochem Biophys Res Commun. 2021 Jan 06;539:48-55 Authors: Oh SH, Lee HJ, Ahn MK, Jeon MY, Yoon JS, Jung YJ, Kim GN, Baek IJ, Kim I, Kim KM, Sung YH 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 m...
Source: Biochemical and Biophysical Research communications - Category: Biochemistry Authors: Tags: Biochem Biophys Res Commun Source Type: research