Bridge Helix of Cas9 Modulates Target DNA Cleavage and Mismatch Tolerance.
Kesavan BabuNadia AmraniWei JiangS D YogeshaRichard NguyenPeter Z QinRakhi RajanPublished in: Biochemistry (2019)
CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection for bacteria and archaea against intruding genomic materials. The programmable nature of CRISPR-targeting mechanisms has enabled their adaptation as powerful genome engineering tools. Cas9, a type II CRISPR effector protein, has been widely used for gene-editing applications owing to the fact that a single-guide RNA can direct Cas9 to cleave desired genomic targets. An understanding of the role of different domains of the protein and guide RNA-induced conformational changes of Cas9 in selecting target DNA has been and continues to enable development of Cas9 variants with reduced off-targeting effects. It has been previously established that an arginine-rich bridge helix (BH) present in Cas9 is critical for its activity. In the present study, we show that two proline substitutions within a loop region of the BH of Streptococcus pyogenes Cas9 impair the DNA cleavage activity by accumulating nicked products and reducing target DNA linearization. This in turn imparts a higher selectivity in DNA targeting. We discuss the probable mechanisms by which the BH-loop contributes to target DNA recognition.
Keyphrases
- crispr cas
- genome editing
- circulating tumor
- single molecule
- cell free
- nucleic acid
- copy number
- circulating tumor cells
- nitric oxide
- protein protein
- cancer therapy
- transcription factor
- genome wide
- gene expression
- oxidative stress
- escherichia coli
- candida albicans
- living cells
- amino acid
- cystic fibrosis
- endothelial cells
- biofilm formation
- regulatory t cells
- molecular dynamics simulations