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Gene-editing technology has revolutionized molecular therapeutics, enabling DNA-engineering-based approaches to treat disease. Despite this, development of medicines using gene editing has been hampered by technological, immunological, and legal limitations. We described previously the discovery of novel type II and type V gene-editing systems from metagenomic data, the characterization of these systems in vitro, and a demonstration of their activity in immortalized cell lines. Here, we substantially advance this work with three separate, novel gene-editing systems, demonstrating their utility for cell therapy development. We express and purify the nuclease components of both type II and type V effectors and show that all three systems are capable of reproducible, high-frequency gene editing in primary immune cells. In human T cells, disruption of the T cell receptor (TCR) alpha-chain constant region occurred in up to 95% of cells, both copies of the TCR beta-chain constant region in up to ~90% of cells, and beta-2 microglobulin in up to 95% of cells. Simultaneous double knock-out of TRAC and TRBC was obtained at an equal frequency. Gene editing with our systems had no effect on T cell viability. Further, we use our novel gene-editing systems to exploit homology-dependent DNA repair to integrate a CAR construct into the TCR alpha-chain locus (in up to ~60% of T cells), and demonstrate high-level CAR expression and antigen-dependent CAR-T cytotoxicity. Such robust editing activity at the TCR loci will permit efficient engineering and manufacture of allogeneic chimeric antigen receptor- (CAR) and TCR-based cell therapies. We next applied our novel gene-editing tools to NK cells and B cells. We achieved almost 100% gene disruption at the CD38 locus in NK cells and integrated a chimeric antigen receptor into the NK cell genome at a frequency of ~40%. Such CAR-NK cells displayed robust CAR-directed cellular cytotoxicity. B cell editing occurred in approximately 80% of target cells with successful transgene integration. Cas9 is notorious for tolerating mismatches and bulges in its target, resulting in high levels of unwanted DNA double-strand break formation. In contrast, interrogation of our gene editing systems using GUIDE-seq reveals no or very few off-target sites even at doses greatly in excess of those required for high-frequency gene editing. Finally, as our systems are taken from microbes found in environmental samples rather than from human pathogens, we expect pre-existing immunity to our nucleases will be quite rare. In all, we show that three new gene-editing systems have the activity, specificity, and translatability necessary for use in cell therapy development. Disclosures No relevant conflicts of interest to declare.