Highly efficient ‘hit-and-run’ genome editing with unconcentrated lentivectors carrying Vpr.Prot.Cas9 protein produced from RRE-containing transcripts

Abstract

<jats:title>Abstract</jats:title> <jats:p>The application of gene-editing technology is currently limited by the lack of safe and efficient methods to deliver RNA-guided endonucleases to target cells. We engineered lentivirus-based nanoparticles to co-package the U6-sgRNA template and the CRISPR-associated protein 9 (Cas9) fused with a virion-targeted protein Vpr (Vpr.Prot.Cas9), for simultaneous delivery to cells. Equal spatiotemporal control of the vpr.prot.cas9 and gag/pol gene expression (the presence of Rev responsive element, RRE) greatly enhanced the encapsidation of the fusion protein and resulted in the production of highly efficient lentivector nanoparticles. Transduction of the unconcentrated, Vpr.Prot.Cas9-containing vectors led to &amp;gt;98% disruption of the EGFP gene in reporter HEK293-EGFP cells with minimal cytotoxicity. Furthermore, we detected indels in the targeted endogenous loci at frequencies of up to 100% in cell lines derived from lymphocytes and monocytes and up to 15% in primary CD4+ T cells by high-throughput sequencing. This approach may provide a platform for the efficient, dose-controlled and tissue-specific delivery of genome editing enzymes to cells and it may be suitable for simultaneous endogenous gene disruption and a transgene delivery.</jats:p>

Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins

“…The results from our study highlight the potential therapeutic benefit of using rational engineering to further advance delivery platforms for gene editing agents. While VLPs have been used previously to deliver Cas9 nuclease RNPs (Campbell et al, 2019;Choi et al, 2016;Gee et al, 2020;Hamilton et al, 2021;Indikova and Indik, 2020;Lyu et al, 2019;Mangeot et al, 2019), and a recent study used VLPs to deliver BE RNPs to HEK293T cells with lower efficiencies than the eVLPs described here (Lyu et al, 2021), no previous study to our knowledge has reported therapeutic levels of postnatal in vivo gene editing of any type using RNP-delivering VLPs. The eVLP platform developed in this work uses a rationally engineered architecture that we customized to package increased amounts of cargo and improve particle titers.…”

Baboon Envelope Pseudotyped “Nanoblades” Carrying Cas9/gRNA Complexes Allow Efficient Genome Editing in Human T, B, and CD34+ Cells and Knock-in of AAV6-Encoded Donor DNA in CD34+ Cells

“…Using chemical induced dimerization Cas9 protein and the gRNAs encoding construct carrying a viral packaging signal both were incorporated into these nanovesicles. Indikova and Indik ( 2020 ) engineered lentivirus-based nanoparticles to co-package the U6-sgRNA template and the CRISPR/Cas9 fused with a virion-targeted protein Vpr (Vpr.Prot.Cas9), for simultaneous delivery to cells. The three systems were highly efficient for gene editing in cell lines and some in primary cells such as induced pluripotent stem cells.…”

Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering

“…This protein fusion orientation allowed guide RNA to associate with fused Cas9 within the MLV VLPs, thereby allowing for the delivery of pre-formed Cas9 RNP complexes capable of mediating robust levels of genome editing in target cells. Recently it has been demonstrated that fusing Cas9 to the lentiviral accessory protein Vpr also promotes Cas9 packaging in budding particles ( Indikova and Indik, 2020 ). Vpr-Cas9-containing lentivirus was highly effective at mediating genome editing in immortalized cell lines but only modestly effective (2.7%–15% indels) at mediating genome editing in primary human CD4 + T cells.…”