Hit and go CAS9 delivered through a lentiviral based self-limiting circuit

Abstract

In vivo application of the CRISPR-Cas9 technology is still limited by unwanted Cas9 genomic cleavages. Long-term expression of Cas9 increases the number of genomic loci non-specifically cleaved by the nuclease. Here we develop a Self-Limiting Cas9 circuit for Enhanced Safety and specificity (SLiCES) which consists of an expression unit for Streptococcus pyogenes Cas9 (SpCas9), a self-targeting sgRNA and a second sgRNA targeting a chosen genomic locus. The self-limiting circuit results in increased genome editing specificity by controlling Cas9 levels. For its in vivo utilization, we next integrate SLiCES into a lentiviral delivery system (lentiSLiCES) via circuit inhibition to achieve viral particle production. Upon delivery into target cells, the lentiSLiCES circuit switches on to edit the intended genomic locus while simultaneously stepping up its own neutralization through SpCas9 inactivation. By preserving target cells from residual nuclease activity, our hit and go system increases safety margins for genome editing.

Self-cutting and integrating CRISPR plasmids (SCIPs) enable targeted genomic integration of genetic payloads for rapid cell engineering

“…Other relatively straightforward AdV (Jin et al 2019;Palmer et al 2020) and other viral (Mangeot et al 2019;Petris et al 2017) methods for gene knock-in have also been described. A single-component…”

“…While particle production required transfecting HEK cells with four separate plasmids, including a fit-for-purpose sgRNA plasmid, particles for HDR-mediated knock-in were generated by complexing them with a synthetic ssDNA oligomer, thereby resulting in successful genome editing in HEK cells and iPSCs (Mangeot et al 2019). Finally, intending to regulate Cas9 levels and function, Petris et al described a Cas9 expression plasmid containing a Cas9-targeting sgRNA with reduced off-target editing effects upon HEK transfection (Petris et al 2017). After transferring this expression construct into a Tet-ON lentiviral vector with the same Cas9-intron which we employed in this study to allow plasmid production, the resulting lentiviral particles limited Cas9 expression levels and enabled equally efficient indel formation as normal Cas9 lentiviruses but with reduced off-target DNA cleavage (Petris et al 2017).…”

“…Inserting an intron into the Cas9 sequence significantly improves self-cutting plasmid stability and production in bacteria We theorized this instability was due to leaky Cas9 and gRNA production in the DH5α cells used for molecular cloning and plasmid production, which would cause plasmid degradation (Figure 2A). To prevent processing of these transcripts in bacteria cells, we inserted a mammalian intron into the Cas9 plasmid genetic sequence, a strategy previously used to increase the production of cleavable CRISPR components (Petris et al 2017) (pQCi; Figure 2B and Supplemental Figure 1B). Plasmids targeting three different sites of the human TRAC gene were created with guide sequences, bait sequences, or both, in pQC and pQCi backbones (generating the 8 plasmid formats in Supplemental Figure 1B).…”

Genetic reprogramming for NK cell cancer immunotherapy with CRISPR/Cas9

“…19 With the rapid development of this technology, designs can be made for recognition of specific loci in the genome to create DNA double-strand breaks (DSBs), which leads to repair and subsequent gene knock-out/knock-in or forced expression. To achieve the delivery, integrating (lentivirus and retrovirus) 21 or non-integrating (adenovirus and adeno-associated virus) [22][23][24] transduction are both applicable approaches. In some cases where gene knock-in is intended, a donor DNA with homology to the sequences flanking the DSB location is required in such a situation.…”

“…NK cells, particularly primary NK cells, are well known to resist ordinary transfection, making the delivery of the Cas9 system difficult, and thus representing an important concern in gene-editing of NK cell immunotherapy. To achieve the delivery, integrating (lentivirus and retrovirus) 21 or non-integrating (adenovirus and adeno-associated virus) [22][23][24] transduction are both applicable approaches. Among these viral vectors, adeno-associated virus is currently the preferred vector for CRISPR/Cas9 system components delivery for immunotherapy, and is usually used for in vivo somatic gene delivery due to their low immunogenicity.…”