Decoding Cas9 orthologs using gBlocks Gene Fragments

Esvelt KM, Mali P, et al. (2013) Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.Nat Methods, 10(11):1116–1121. doi: 10.1038/nmeth.2681.

Citation summary: Researchers use gBlocks® Gene Fragments to create Cas9 orthologs as well as tracRNA expression cassettes for each ortholog tested. Read how.

Jan 15, 2014

Cas9 derived from Streptococcus pyogenes has been widely used for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) mediated genome editing. This is due in part to the short NGG sequence requirement of the protospacer adjacent motif (PAM) sequence requirements of S pyogenes Cas9. The authors of this study use IDT gBlocks Gene Fragments to generate human optimized Cas9 orthologs from S thermophilus, N meningitidis, and T denticola, as well as tracRNA expression cassettes for each ortholog.

The authors provide new information about the recognition patterns and other characteristics of the Cas9 enzymes. They also demonstrate a possible scenario for use of more than one Cas9, by combining Cas9 orthologs with different PAM requirements to carry out parallel tasks in bacterial and human cell lines.