The CRISPR-Cas12a Platform for Accurate Genome Editing, Gene Disruption, and Efficient Transgene Integration in Human Immune Cells

Abstract

CRISPR-Cas12a nucleases have expanded the toolbox for targeted genome engineering in a broad range of organisms. Here, using a high-throughput engineering approach, we explored the potential of a novel CRISPR-MAD7 system for genome editing in human cells. We evaluated several thousand optimization conditions and demonstrated accurate genome reprogramming with modified MAD7. We identified crRNAs that allow for ≤95% non-homologous end joining (NHEJ) and 66% frameshift mutations in various genes and observed the high-cleavage fidelity of MAD7 resulting in undetectable off-target activity. We explored the dsDNA delivery efficiency of CRISPR-MAD7, and by using our optimized transfection protocol, we obtained ≤85% chimeric antigen receptor (CAR) insertions in primary T cells, thus exceeding the baseline integration efficiencies of therapeutically relevant transgenes using currently available virus-free technologies. Finally, we evaluated multiplex editing efficiency with CRISPR-MAD7 and demonstrated simultaneous ≤35% CAR transgene insertions and ≤80% gene disruption efficiencies. Both the platform and our transfection procedure are easily adaptable for further preclinical studies and could potentially be used for clinical manufacturing of CAR T cells.

Engineered yeast cells simulating CD19+ cancers to control CAR T cell activation

“…For this purpose, we proceeded to demonstrate the clinical relevance of applying SCASA yeast cells in characterizing a donor-derived CAR T cell product’s performance. As a case study, we applied a novel CRISPR-MAD7 method for CAR transgene insertion via electroporation in pan-T cells isolated from a healthy donor 73 , to generate an alternative Hu19-CD8ɑ-CD28-CD3ζ CAR T cell product containing fully- human antigen-binding domains 74,75 . The response profiles and population behavior of this CAR T cell product were characterized by flow cytometry after co-cultivation with SCASA yeast cell design P PGK1 - CD19 with direct comparison to NALM6 cancer cells at three different cell-to-cell ratios; 0.2x, 1.0x, and 5.0x target cells to 100,000 alive CAR T cells.…”

“…T cells were then resuspended at 1×10 6 cells/mL in ImmunoCult™-XF T Cell Expansion Medium (Stemcell Technologies, Cat.#10981) supplemented with Human Recombinant IL-2 (12.5 ng/mL) (Stemcell Technologies, Cat.#78036), IL-7 (5 ng/mL) (Stemcell Technologies, Cat.#78053), IL-15 (5 ng/mL) (Stemcell Technologies, Cat.#78031), and ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (25 µL/mL) (Stemcell Technologies, Cat.#10990) and cultured for 1.5 days at 37°C with 5% CO 2 . After activation, T cells were engineered using CRISPR-MAD7 according to a recently published method, 73 to insert the CAR Hu19-CD8ɑ-CD28-CD3ζ (Hu19-CD828Z), containing an anti-CD19 fully-human scFv (Hu19), CD8ɑ hinge and transmembrane domains, a CD28 costimulatory domain, and a CD3ζ activation domain 74 (Clinical trial: NCT02659943). In addition, the CAR contained a myc-tag for the detection of surface expression.…”

“…The CAR was expressed using the EF-1ɑ promoter and a bovine growth hormone polyadenylation (bgh-PolyA) signal from the AAVS1 safe-harbor site. The CRISPR- MAD7 crRNA ribonucleoprotein formulation, Hu19-CD8ɑ-CD28-CD3ζ homology-directed repair (HDR) template generation, and transfection of donor-derived (primary) T cells using a Lonza 4D- Nucleofector with shuttle unit with use of M3814 (Selleckchem) recovery followed the previously described methods 73 . Along with the CAR T cells, a control (CTRL) T cell product was established from the same T cell isolate, however without using a CAR HDR template.…”

“…: ACC 128) was included as a benchmark positive control. NALM6 was cultured in RPMI+10%FBS+1%PS at 37°C with 5% The cell line was further engineered by genomic insertion of a CAG-promoter driven GFPcassette in safe-harbor site AAVS1 using CRISPR-MAD7, as previously described 73 .…”

“…Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coat, by blood filtration using a 25µL/mL) (Stemcell Technologies, Cat.#10990) and cultured for 1.5 days at 37°C with 5% CO2. After activation, T cells were engineered using CRISPR-MAD7 according to a recently published method, 73 to insert the CAR Hu19-CD8ɑ-CD28-CD3ζ (Hu19-CD828Z), containing an anti-CD19 fully-human scFv (Hu19), CD8ɑ hinge and transmembrane domains, a CD28 costimulatory domain, and a CD3ζ activation domain 74 (Clinical trial: NCT02659943). In addition, the CAR contained a myc-tag for the detection of surface expression.…”