Detecting non-canonically-targeted SpCas9 generated DSBs in human cells by LAM-HTGTS
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ABSTRACT:
PROVIDER: PRJNA791602 | ENA |
REPOSITORIES: ENA
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Project description:Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility of CRISPR technology. Here, we benchmark PAM preferences, on-target activity, and off-target susceptibility of 11 variants of SpCas9 in cell culture assays with thousands of guides targeting endogenous genes. To enhance the coverage and thus utility of base editing screens, we demonstrate that the SpCas9-NG and SpG variants are compatible with both A>G and C>T base editors, more than tripling the number of guides and assayable residues. We demonstrate the performance of these technologies by screening for loss-of-function mutations in BRCA1 and Venetoclax-resistant mutations of BCL2, identifying both known and new insights into these clinically-relevant genes. We anticipate that the tools and methodologies described here will facilitate the investigation of genetic variants at a finer and deeper resolution for any locus of interest.
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Project description:High-throughput genome-wide translocation sequencing (HTGTS) is a robust approach to identify genome-wide translocation junctions. We performed HTGTS to study the fate of introduced c-myc DSBs in mouse splenic B cells activated for activation cytidine deaminase (AID)-dependent class switch recombination (CSR). We found frequent translocations of c-myc DSBs to AID-initiated DSBs in IgH switch regions in wild-type (WT) and ATM-deficient B cells. However, c-myc also translocated frequently to newly generated DSBs within a 35-megabase region downstream of IgH in ATM-deficient, but not WT, CSR-activated B cells. Moreover, we found such DSBs and translocations in activated B cells that did not express AID or undergo CSR. These findings indicate that ATM deficiency leads to formation of chromosome 12 dicentrics via RAG-initiated IgH DSBs in progenitor B cells and that these dicentrics can be propagated developmentally into mature B cells where they generate new DSBs downstream of IgH via breakage-fusion-bridge cycles.
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