PAM-flexible dual base editor-mediated random mutagenesis and self-activation strategies to improve CRISPRa potency.

ABSTRACT: VP64 is the smallest transactivation domain that can be packaged together with the sgRNA into a single adeno-associated virus (AAV) vector. However, VP64-based CRISPRa often exerts modest activation to the target gene when only one sgRNA is used. Herein, we used PAM-flexible dual base editor-mediated mutagenesis and self-activation strategies to derive VP64 variants with gain-of-function mutations. First, we generated an HEK293FT transgenic clone to stably expressing pTK-CRISPRa-GFP. The sgRNA of CRISPRa was designed to target the TK promoter, thereby allowing self-activation of CRISPRa-GFP. Base editors were then used to randomly mutagenesis VP64 in this transgenic cell. VP64 with enhanced potency would translate into increment of GFP fluorescence intensity, thereby allowing positive sele

SUBMITTER: Lau CH 

PROVIDER: S-EPMC9198377 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature