Directed evolution studies of a thermophilic Type II-C Cas9

• Published in: Methods in enzymology Vol. 616; p. 265
• Main Authors: Hand, Travis H, Das, Anuska, Li, Hong
• Format: Journal Article
• Language: English
• Published: United States 01-01-2019
• Subjects: Actinobacteria - chemistry; Actinobacteria - genetics; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; CRISPR-Associated Protein 9 - chemistry; CRISPR-Associated Protein 9 - genetics; Directed Molecular Evolution - methods; Gene Library; Models, Molecular; Cas9; Off-targets; Thermophilic Cas9; PAM; Type II-C CRISPR–Cas
• ISSN: 1557-7988
• DOI: 10.1016/bs.mie.2018.10.029

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.