A Cas9 nanoparticle system with truncated Cas9 target sequences on DNA repair templates enhances genome targeting in diverse human immune cell types

Abstract

~150 words and referenced]Virus-modified T cells are approved for cancer immunotherapy, but more versatile and precise genome modifications are needed for a wider range of adoptive cellular therapies 1-4 . We recently developed a non-viral CRISPR-Cas9 system for genomic site-specific integration of large DNA sequences in primary human T cells 5 . Here, we report two key improvements for efficiency and viability in an expanded variety of clinically-relevant primary cell types. We discovered that addition of truncated Cas9 target sequences (tCTS) at the ends of the homology directed repair (HDR) templates can interact with Cas9 ribonucleoproteins (RNPs) to 'shuttle' the template and enhance targeting efficiency. Further, stabilizing the Cas9 RNPs into nanoparticles with poly(glutamic acid) improved editing, reduced toxicity, and enabled lyophilized storage without loss of activity. Combining the tCTS HDR template modifications with polymer-stabilized nanoparticles increased gene targeting efficiency and viable cell yield across multiple genomic loci in diverse cell types. This system is an inexpensive, user-friendly delivery platform for nonviral genome reprogramming that we successfully applied in regulatory T cells (Tregs), -T cells, B cells, NK cells, and primary and iPS-derived 6 hematopoietic stem progenitor cells (HSPCs).We recently reported an approach to reprogram human T cells with CRISPR-based genome targeting without the need for viral vectors 5 . However, many research and clinical applications still depend upon improved efficiency, cell viability, and generalizability of non-viral genome targeting across cell types 1-4 . We previously found that varying the relative concentrations of both Cas9 RNP and HDR template had significant effects on targeting efficiency

Rapid discovery of synthetic DNA sequences to rewrite endogenous T cell circuits

“…We recently developed an efficient non-viral genome targeting method to modify endogenous genes in primary human T cells 6,7 . Unlike non-targeted gene transduction with viral vectors, CRISPR-based knock-in requires a combination of gRNA and homology-directed repair (HDR) template sequences to target a transgenic DNA sequence to a specified genomic site.…”

“…CRISPR technology has transformed our ability to manipulate the human genome in therapeutically-relevant cell types 5–7 . High-throughput screening methods are necessary to explore the infinite number of potential manipulations possible for therapeutic relevance.…”

“…To produce RNPs, the crRNA and tracrRNA aliquots were thawed, mixed 1:1 by volume, and annealed by incubation at 37□ °C for 30 min to form an 80 µM gRNA solution. Next, PGA mixed with freshly-prepared gRNA at 0.8:1 volume ratio prior to complexing with Cas9 protein for final volume ratio gRNA:PGA:Cas9 of 1:0.8:1 (2:1 gRNA to Cas9 molar ratio) and incubated at 37□ °C for 15 min to form a 14.3 µM RNP solution 6,7 . RNPs were electroporated immediately after complexing.…”

“…1 ), HDRTs were first aliquoted into wells of a 96-well polypropylene V-bottom plate. Poly Glutamic Acid was added between the gRNA and Cas9 complexing step during RNP assembly as described 7 . Complexed RNPs were then added to the HDRTs and allowed to incubate together at room temperature for at least 30s.…”

“…For GEEPs knock-ins and pooled knock-in screens ( Fig. 2-4 ), truncated Cas9 Target Sequences (tCTS) were additionally added to the 5’ and 3’ ends of the HDR template enabling a Cas9 ‘shuttle’ as described 7 . For all variations, T cells resuspended in the electroporation buffer were added to the RNP and HDRT mixture, briefly mixed, and then transferred into a 96-well electroporation cuvette plate…”