Directed evolution studies of a thermophilic Type II-C Cas9

Abstract

Though making up nearly half of the known CRISPR–Cas9 family of enzymes, the Type II-C CRISPR–Cas9 has been underexplored for their molecular mechanisms and potential in safe gene editing applications. In comparison with the more popular Type II-A CRISPR–Cas9, the Type II-C enzymes are generally smaller in size and utilize longer base pairing in identification of their DNA substrates. These characteristics suggest easier portability and potentially less off-targets for Type II-C in gene editing applications. We describe identification and biochemical characterization of a thermophilic Type II-C CRISPR–Cas from Acidothermus cellulolyticus (AceCas9). We describe several library-based methods that enabled us to identify the PAM sequence and elements critical to protospacer mismatch surveillance of AceCas9

The molecular basis for recognition of 5′-NNNCC-3′ PAM and its methylation state by Acidothermus cellulolyticus Cas9

“…Plasmid library preparation. Libraries used for directed protein or RNA evolution were prepared as previously described 31 . Briefly, targeted locations were amplified with Gibson Assembly primers (Table S3) via error-prone PCR (to produce an average of 1.5 mutation per 100 bp) and inserted back into the pACYCDuet-Ac9-g106 vector 29 .…”

Catalytically Enhanced Cas9 through Directed Protein Evolution

“…The survival assay was carried out as previously described [34][35][36] . Briefly, electrocompetent E. coli BW25141 containing the modified p11-LacY-wtx1 (ccdB encoding) plasmid were transformed with 100-200 ng of AceCas9 library plasmids.…”

“…Plasmid cleavage assays were performed as previously described [34][35][36] . Briefly, 500 nM pre-annealed AceCas9: sgRNA complex was incubated with 6 nM plasmid DNA in cleavage buffer (20 mM Tris pH 7.5, 150 mM KCl, 2 mM DTT, 10 mM MgCl2, 5% glycerol) at 50 °C for a given time.…”

“…Mutations that disrupt one of the two hinge helices lowered the DNA cleavage activity of SpyCas9 27 .These detailed mechanistic studies suggest that the allosteric hinge is also a potential target for engineering CECas9 33 . However, protein engineering by rational design to select for CECas9 variants would be laborious and have limited chances of successes.We previously described a toxicity-based bacterial survival assay that is amenable for a library-based directed protein evolution selection 34,35 . In this approach, a library of plasmids encoding a collection of Cas9 variants is transformed into bacterial cells harboring a plasmid expressing the toxic protein ccdB under the induction by arabinose.…”

“…We previously described a toxicity-based bacterial survival assay that is amenable for a library-based directed protein evolution selection 34,35 . In this approach, a library of plasmids encoding a collection of Cas9 variants is transformed into bacterial cells harboring a plasmid expressing the toxic protein ccdB under the induction by arabinose.…”