Enhancing CRISPRCas9 Genome Editing and Lentiviral Gene Delivery to Defined Cells Implications for Therapeutics and Diagnostics

Abstract

Clustered-Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR newlineassociated (Cas) proteins (CRISPR/Cas) were identified initially in prokaryotes as newlineadaptive immunity components. In 2012, the first demonstration of CRISPR/Cas as a newlineprogrammable Dual-RNA Guided DNA Endonuclease highlighted its potential for newlineprecision genome engineering. The Cas protein from Streptococcus pyogenes is used newlinealong with the single-guide RNA (sgRNA) to target the locus to generate a doublestrand newlinebreak (DSB), which is subsequently repaired by the DNA repair pathways in newlinemammalian cells. It has been observed that CRISPR/Cas9 efficiency varies with newlinemammalian cell types and/or the locus being edited, indicating the presence of newlinerepressive mechanisms. To find out, we performed a large-scale screen and identified newlinea competing cellular pathway (TGFand#946; signaling) that represses CRISPR/Cas9-mediated newlineloss-of-function editing, in a manner independent of cell-types or the mode-of Cas9 newlineexpression. We demonstrated that pharmacological inhibition of TGFand#946; signaling or newlinegenetic ablation of TGFand#946; receptor promotes CRISPR/Cas9 editing efficiency in various newlinecell lines used in biomedical research and primary human cells of clinical relevance. newlineThese findings have potential implications for therapeutic genome editing and the newlinegeneration of CRISPR/Cas9-based disease models. newlineLentiviral vectors are effectively used to deliver CRISPR/Cas9 components to newlinemammalian cells for gene-editing. These vectors are also useful reporters when newlinepseudotyped with different envelopes to evaluate viral entry inhibitors. The infection newlineby SARS-CoV-2 in human cells represents an example where the virus envelope newlineglycoprotein (spike) recognizes and infects ACE2 expressing cells. While the study of newlinelive SARS-CoV-2 necessitates high-containment settings, spike pseudotyped lentiviral newlinereporter vectors present a viable alternative. Accordingly, we pseudotyped lentiviral newlinevector using SARS-CoV-2 spike glycoprotein to target human ACE2-expressing cells. newlineThis approach not only enabled the identification of SARS-CoV-2 nucleoprotein as newlinevirion infectivity enhancer but also offered a robust high-throughput platform for newlineserodiagnosis. Using this serodiagnosis method developed in-house, we uncovered newlinemolecular determinants of the SARS-CoV-2 spike that underlie the SARS-CoV-2 newlinevariant s ability to cause breakthrough infections. newline