Analysis of Wild Type LbCpf1 Protein, and PAM Recognition Variants, in a Cellular Context.

ABSTRACT: Nucleases used in genome engineering induce hydrolysis of DNA phosphate backbone in a sequence-specific manner. So far CRISPR-Cas, the RNA-guided nucleases, is the most advanced genome engineering system. The CRISPR nucleases allows recognition of a particular genomic sequence with two distinct molecular interactions: first, by direct interaction between the nuclease and the protospacer-adjacent motif, wherein discrete amino acids interact with DNA base pairs; and second, by hybridization of the guide RNA with the target DNA sequence. Here we report the application of the single strand annealing cellular assay to analyze and quantify nuclease activity of wild type and mutant CRISPR-Cpf1. Using this heterologous marker system based on GFP activity, we observed a comparable PAM recognition s

SUBMITTER: Shin U 

PROVIDER: S-EPMC7817983 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature