Bridge Helix of Cas9 Impacts Target DNA Cleavage

Abstract

CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection for bacteria and archaea against intruding genomic materials. Cas9, a type II CRISPR effector protein, is widely used for gene editing applications since a single guide RNA (sgRNA) can direct Cas9 to cleave DNA targets of interest. The complementary base pairing between the guide region of the sgRNA and the target DNA is essential to activate the endonuclease sites of Cas9. In addition, a three to five nucleotide long region, termed protospacer adjacent motif (PAM), adjacent to the RNA-DNA complementary region is essential for Cas9 function. The mechanism of RNA-mediated conformational changes in Cas9 and how they contribute to faithful DNA targeting is being pursued actively for developing Cas9 variants with minimal 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 substitutions within the BH of Streptococcus pyogenes (Spy) Cas9 impair DNA cleavage activity compared to the wild-type protein. The DNA cleavage deficiency is more pronounced in DNA targets that are mismatched with the guide region of sgRNA at the PAM proximal side. BH substitutions cause an accumulation nicked products, leading to a reduction of target DNA linearization. Gene editing experiments performed on human cells (HEK293T) showed that even though the BH-variant is not as efficient as the wild-type protein in editing all the tested sites, the off-target effect on edited sites is considerably reduced when compared to the wild-type protein. Mechanistic analyses show that the BH-substitution does not reduce sgRNA-binding affinity, but substantially reduces the stability of the protein-RNA complex. We propose BH-substitution as a mechanism to fine-tune CRISPR-based gene editing applications since BH is conserved in several CRISPR systems.