Guide RNA scaffold variants enabled easy cloning of large gRNA cluster for multiplexed gene editing.

ABSTRACT: Cas9 protein-mediated gene editing has revolutionized genetic manipulation in most organisms. There are many cases where multiplexed gene editing is needed. Cas9 is capable of multiplex gene editing when expressed with multiple guide RNAs. Conventional cloning methods for multiplexed gene editing vector is not efficient due to repeated use of a single-guide RNA scaffold and inefficient ligation. In this study, we conducted structure-guided mutagenesis and random mutagenesis on the original sgRNA scaffold and identified a large number of functional sgRNA scaffold variants. With these scaffold variants and different tRNAs, fusion polymerase chain reaction protocol was developed to rapidly synthesize spacer-scaffold-tRNA-spacer units with up to 9 targets. In conjunction with golden gate cloni

SUBMITTER: Wang Y 

PROVIDER: S-EPMC10826992 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature