Abstract
The mechanism underlying unwanted structural variations induced by CRISPR-Cas9 remains poorly understood, and no effective strategy is available to inhibit the generation of these byproducts. Here we find that the generation of a high level of translocations is dependent on repeated cleavage at the Cas9-targeting sites. Therefore, we employ a strategy in which Cas9 is fused with optimized TREX2 to generate Cas9TX, a Cas9 exo-endonuclease, which prevents perfect DNA repair and thereby avoids repeated cleavage. In comparison with CRISPR-Cas9, CRISPR-Cas9TX greatly suppressed translocation levels and enhanced the editing efficiency of single-site editing. The number of large deletions associated with Cas9TX was also reduced to very low level. The application of CRISPR-Cas9TX for multiplex gene editing in chimeric antigen receptor T cells nearly eliminated deleterious chromosomal translocations. We report the mechanism underlying translocations induced by Cas9, and propose a general strategy for reducing chromosomal abnormalities induced by CRISPR-RNA-guided endonucleases.
Highlights
The mechanism underlying unwanted structural variations induced by CRISPR-Cas[9] remains poorly understood, and no effective strategy is available to inhibit the generation of these byproducts
We identify a large number of chromosomal translocations in T cells targeted at the TRAC, TRBC, and PDCD1 genes on day 3 post-editing, and some of them are still present on days 7 and 14
In the primer extension-mediated sequencing (PEM-seq) data, the ratio of indels to total sequencing junctions is defined as the editing efficiency, while the ratio of translocations to indels plus translocations is defined as the percentage of translocations
Summary
The mechanism underlying unwanted structural variations induced by CRISPR-Cas[9] remains poorly understood, and no effective strategy is available to inhibit the generation of these byproducts. We report the mechanism underlying translocations induced by Cas[9], and propose a general strategy for reducing chromosomal abnormalities induced by CRISPR-RNA-guided endonucleases. In addition to insertions and deletions (indels), Streptococcus pyogenes Cas[9] (SpCas9) nuclease has been reported to lead to unwanted structural variations, including chromosomal translocations, large deletions, and other complex rearrangements[6–10]. The concern regarding translocations is exacerbated in multiplex gene editing[10,15,16,21] In this context, several genes involved in alloreactivity (TRAC, TRBC, and B2M) and immunosuppression (PDCD1) are simultaneously disrupted by SpCas[9] nuclease to optimize chimeric antigen receptor (CAR) T cell therapy and T cell receptor (TCR) T cell therapy. In order to reduce the number of translocations, we propose a general strategy in which an exonuclease domain is fused to SpCas[9] to generate an exo-endonuclease form and prevent repeated cleavage during multiplex genome editing. Our findings provide a general method for the application of engineered sequencespecific nucleases to reduce the tendency for chromosomal abnormalities
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