RESUMEN
Guided by the extensive knowledge of CRISPR-Cas9 molecular mechanisms, protein engineering can be an effective method in improving CRISPR-Cas9 toward desired traits different from those of their natural forms. Here, we describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9) by targeting a shortened protospacer within a toxic gene. We demonstrate the effectiveness of this method with a previously characterized Type II-C Cas9 from Acidothermus cellulolyticus (AceCas9) and show by enzyme kinetics an up to fourfold improvement of the in vitro catalytic efficiency by AceCECas9. We further evolved the more widely used Streptococcus pyogenes Cas9 (SpyCas9) and demonstrated a noticeable improvement in the SpyCECas9-facilitated homology directed repair-based gene insertion in human colon cancer cells.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Ingeniería de Proteínas , Actinobacteria/enzimología , Actinobacteria/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Neoplasias del Colon , Edición Génica/métodos , Células HCT116 , Humanos , Streptococcus pyogenes/enzimología , Streptococcus pyogenes/genéticaRESUMEN
Acidothermus cellulolyticus CRISPR-Cas9 (AceCas9) is a thermophilic Type II-C enzyme that has potential genome editing applications in extreme environments. It cleaves DNA with a 5'-NNNCC-3' Protospacer Adjacent Motif (PAM) and is sensitive to its methylation status. To understand the molecular basis for the high specificity of AceCas9 for its PAM, we determined two crystal structures of AceCas9 lacking its HNH domain (AceCas9-ΔHNH) bound with a single guide RNA and DNA substrates, one with the correct and the other with an incorrect PAM. Three residues, Glu1044, Arg1088, Arg1091, form an intricate hydrogen bond network with the first cytosine and the two opposing guanine nucleotides to confer specificity. Methylation of the first but not the second cytosine base abolishes AceCas9 activity, consistent with the observed PAM recognition pattern. The high sensitivity of AceCas9 to the modified cytosine makes it a potential device for detecting epigenomic changes in genomes.
Asunto(s)
Actinobacteria/enzimología , Proteína 9 Asociada a CRISPR/química , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Edición Génica/métodos , Actinobacteria/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Cristalografía por Rayos X , Citosina , ADN/química , ADN/genética , ADN/metabolismo , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento , Metilación , Modelos Moleculares , Conformación Proteica , ARN Guía de Kinetoplastida/químicaRESUMEN
Despite being utilized widely in genome sciences, CRISPR-Cas9 remains limited in achieving high fidelity in cleaving DNA. A better understanding of the molecular basis of Cas9 holds the key to improve Cas9-based tools. We employed direct evolution and in vitro characterizations to explore structural parameters that impact the specificity of the thermophilic Cas9 from Acidothermus cellulolyticus (AceCas9). By identifying variants that are able to cleave mismatched protospacers within the seed region, we found a critical role of the phosphate lock residues in substrate specificity in a manner that depends on their sizes and charges. Removal of the negative charge from the phosphate lock residues significantly decreases sensitivity to the guide-DNA mismatches. An increase in size of the substituted residues further reduces the sensitivity to mismatches at the first position of the protospacer. Our findings identify the phosphate lock residues as an important site for tuning the specificity and catalytic efficiency of Cas9.
