Utilizing directed evolution to interrogate and optimize CRISPR/Cas guide RNA scaffolds.

ABSTRACT: CRISPR-based editing has revolutionized genome engineering despite the observation that many DNA sequences remain challenging to target. Unproductive interactions formed between the single guide RNA's (sgRNA) Cas9-binding scaffold domain and DNA-binding antisense domain are often responsible for such limited editing resolution. To bypass this limitation, we develop a functional SELEX (systematic evolution of ligands by exponential enrichment) approach, termed BLADE (binding and ligand activated directed evolution), to identify numerous, diverse sgRNA variants that bind Streptococcus pyogenes Cas9 and support DNA cleavage. These variants demonstrate surprising malleability in sgRNA sequence. We also observe that particular variants partner more effectively with specific DNA-binding antisens

SUBMITTER: Bush K 

PROVIDER: S-EPMC10529641 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature