CRISPR-Cas9 Alternatives: Cutting through the Boundaries of Gene Editing
The use of T cells from healthy donors for allogeneic chimeric antigen receptor T (CAR-T) cell cancer therapy is attractive because healthy donor T cells can produce versatile off-the- shelf CAR-T treatments. To maximize safety and durability of allogeneic products, the endogenous T cell receptor and ma-jor histocompatibility complex class I molecules are often removed via knockout of T cell receptor beta constant (TRBC) (or T cell receptor alpha constant [TRAC]) and B2M, respectively. However, gene editing tools (e.g., CRISPR-Cas9)
can display poor fidelity, which may result in dangerous off- target mutations. Additionally, many gene editing technologies require T cell activation, resulting in a low percentage of desirable stem cell memory T cells (TSCM). We characterize an RNA- guided endonuclease, called Cas-CLOVER, consisting of the Clo051 nuclease domain fused with catalytically dead Cas9. In primary T cells from multiple donors, we find that Cas-CLOVER is a high-fidelity site-specific nuclease, with low off-target activity. Notably, Cas-CLOVER yields efficient multiplexed gene editing in resting T cells.
Allogeneic CAR-T therapies have the potential to dramatically increase patient access and decrease production costs for the treatment of a variety of malignancies. The pre-selection of optimal donor pro- files is also a key advantage for allogeneic approaches. However, an effective allogeneic approach is critically dependent on clean and efficient gene editing; product safety hinges on the fidelity of the intended genetic manipulations in T cells.
Here we describe a dimeric, high-fidelity SSN, Cas-CLOVER, which consists of a fusion of catalytically dead SpCas9 (dCas9) with the nuclease domain from a Clostridium Clo051 type IIs restriction endonuclease. This yields a nuclease whose activity is predicated upon the dimerization of the Clo051 nuclease domain, enabled by RNA guided recognition of two adjacent 20-nt target sequences. We find that Cas-CLOVER has low off-target nuclease activity in conjunction with the piggyBac transposon for transgenesis with a CAR specific for the B cell maturation antigen (BCMA), Cas-CLOVER enables the safe production of a fully allogeneic CAR-T product candidate.
https://www.cell.com/molecular-therapy-family/nucleic-acids/fulltext/S2162-2531(22)00155-X
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