A detailed cell-free transcription-translation-based assay to decipher CRISPR protospacer-adjacent motifs.

ABSTRACT: The RNA-guided nucleases derived from the CRISPR-Cas systems in bacteria and archaea have found numerous applications in biotechnology, including genome editing, imaging, and gene regulation. However, the discovery of novel Cas nucleases has outpaced their characterization and subsequent exploitation. A key step in characterizing Cas nucleases is determining which protospacer-adjacent motif (PAM) sequences they recognize. Here, we report advances to an in vitro method based on an E. coli cell-free transcription-translation system (TXTL) to rapidly elucidate PAMs recognized by Cas nucleases. The method obviates the need for cloning Cas nucleases or gRNAs, does not require the purification of protein or RNA, and can be performed in less than a day. To advance our previously published method,

SUBMITTER: Maxwell CS 

PROVIDER: S-EPMC6051895 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature