Large scale screening of CRISPR guide RNAs using an optimized high throughput robotics system.

ABSTRACT: All CRISPR/CAS systems utilize CRISPR guide RNAs (crRNAs), the design of which depend on the type of CAS protein, genetic target and the environment/matrix. While machine learning approaches have recently been developed to optimize some crRNA designs, candidate crRNAs must still be screened for efficacy under relevant conditions. Here, we demonstrate a high-throughput method to screen hundreds of candidate crRNAs for activation of Cas13a collateral RNA cleavage. Entire regions of a model gene transcript (Y. pestis lcrV gene) were tiled to produce overlapping crRNA sets. We tested for possible effects that included crRNA/target sequence, size and secondary structures, and the commercial source of DNA oligomers used to generate crRNAs. Detection of a 981 nt target RNA was initially successfu

SUBMITTER: Spangler JR 

PROVIDER: S-EPMC9385653 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature