Protein engineering of Cas9 for safer genome tools, and phylogenetic analysis of Coronavirus spike protein for efficient antiviral targets

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins are adaptive immune systems that protect bacteria and archaea from mobile genetic elements. These systems have been repurposed for gene editing biotechnologies. CRISPR-Cas9-based gene therapies are in clinical trials, but a safety concern is that Streptococcus pyogenes Cas9 can trigger non-specific DNA damage when not bound to guide RNA (gRNA). Such gRNA-free damage to genomic DNA in human nuclei was reported recently with Streptococcus pyogenes Cas9 (SpyCas9) in human cells transfected with plasmids carrying the Cas9 but devoid of a gRNA, signifying the prevalence of such promiscuous DNA damage under cellular conditions. To eliminate these non-specific cleavage activities, SpyCas9 variants are being developed through our research. One mutation that successfully removes Mn2+-dependent gRNA-free DNA cleavage is SpyCas9H982A. Elucidating the fidelity-increasing DNA cleavage mechanisms of SpyCas9H982A will advance the development of safe Cas9-based gene therapies.

Another safety concern is the finding that SpyCas9 cuts the DNA at unintended sites that partially hybridize to the guide RNA. Effects of such off-targeting include increased risks of cancer and other health problems. The SpyCas9 L64P/K65P substitution variant has been shown to have higher selectivity such that it is less likely to cleave DNA when there is a mismatch to the guide RNA. In this research, a SpyCas9 L64A/K65A variant was constructed and assayed for RNA-guided DNA cleavage to determine whether the loss of intra-protein interactions or compromised structure of the helix containing L64/K65 has the primary role in eliciting this increase in specificity. In vitro DNA cleavage assays showed that the compromised helical structure causes the increase in selectivity for on-target as opposed to PAM-proximal mismatched DNA cleavage in terms of linearization efficiency.

A novel coronavirus outbreak of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) occurred in December 2019, and variants of the virus have been emerging and causing surges in cases, hospitalizations, deaths, and societal disruptions over the past few years. Vaccines have been developed, but research on a variety of vaccines and potentially antiviral targets for small-molecule drugs is helpful for preventing infections from future variants. Initial contact between human cells and SARS-CoV-2 is mediated by the viral Spike (S) protein. The objective of this research is to identify antiviral target sites in S protein and categorize them into sites that are highly evolving and that are highly conserved in a range of human-infecting coronaviruses. The rationale is that targeted drug discovery to these two types of sites will enable better preparedness in tackling coronavirus as it continues to evolve. S protein sequence conservation was analyzed among severe disease causing human coronaviral strains (including MERS, SARS-CoV, and SARS-CoV-2), along with molecular docking of the S protein receptor binding domain with a compound library. Our results identified four pockets and six compounds that are promising for rational drug development.