Description
The Selection of Catalytically Enhanced Cas9 via Protein Evolution The Clustered Regularly Interspaced Palindromic Repeat (CRISPR)-Cas9 system has found widespread applications in genome engineering due to its simplicity and effectiveness. Significant efforts in enzyme engineering have been made to improve the CRISPR-Cas9 systems beyond their natural power by using protein evolution strategies. Relatively less attention, however, has been paid to improving the catalytic efficiency of CRISPR-Cas9. Increased catalytic efficiency may be desired in applications where the currently available CRISPR-Cas9 tools are either ineffective, or of low efficiency such as with the type II-C Cas9. We describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9). We demonstrate the effectiveness of this method with a previously characterized Type II-C Cas9 from Acidothermus cellulolyticus (AceCas9) and the Type II-A Cas9 from Streptococcus pyogenes (SpyCas9).
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