CRISPR-Cas12a-integrated transgenes in genomic safe harbors retain high expression in human hematopoietic iPSC-derived lineages and primary cells.

Discovery of genomic safe harbor sites (SHSs) is fundamental for multiple transgene integrations, such as reporter genes, chimeric antigen receptors (CARs), and safety switches, which are required for safe cell products for regenerative cell therapies and immunotherapies. Here we identified and characterized potential SHS in human cells. Using the CRISPR-MAD7 system, we integrated transgenes at these sites in induced pluripotent stem cells (iPSCs), primary T and natural killer (NK) cells, and Jurkat cell line, and demonstrated efficient and stable expression at these loci. Subsequently, we validated the differentiation potential of engineered iPSC toward CD34+ hematopoietic stem and progenitor cells (HSPCs), lymphoid progenitor cells (LPCs), and NK cells and showed that transgene expression was perpetuated in these lineages. Finally, we demonstrated that engineered iPSC-derived NK cells retained expression of a non-virally integrated anti-CD19 CAR, suggesting that several of the investigated SHSs can be used to engineer cells for adoptive immunotherapies.

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

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.

A Mad7 System for Genetic Engineering of Filamentous Fungi.

The introduction of CRISPR technologies has revolutionized strain engineering in filamentous fungi. However, its use in commercial applications has been hampered by concerns over intellectual property (IP) ownership, and there is a need for implementing Cas nucleases that are not limited by complex IP constraints. One promising candidate in this context is the Mad7 enzyme, and we here present a versatile Mad7-CRISPR vector-set that can be efficiently used for the genetic engineering of four different Aspergillus species: Aspergillus nidulans, A. niger, A. oryzae and A. campestris, the latter being a species that has never previously been genetically engineered. We successfully used Mad7 to introduce unspecific as well as specific template-directed mutations including gene disruptions, gene insertions and gene deletions. Moreover, we demonstrate that both single-stranded oligonucleotides and PCR fragments equipped with short and long targeting sequences can be used for efficient marker-free gene editing. Importantly, our CRISPR/Mad7 system was functional in both non-homologous end-joining (NHEJ) proficient and deficient strains. Therefore, the newly implemented CRISPR/Mad7 was efficient to promote gene deletions and integrations using different types of DNA repair in four different Aspergillus species, resulting in the expansion of CRISPR toolboxes in fungal cell factories.