Min J, Zhao J, Zagelbaum J, Lee J, Takahashi S, Cummings P, Schooley A, Dekker J, Gottesman ME, Rabadan R, Gautier J.

Mol Cell. 2023 Jul 20;83(14):2434-2448.e7. doi: 10.1016/j.molcel.2023.06.016

Mechanisms of insertions at a DNA double-strand break.

Indel-seq: double-strand breaks are induced with the AsiSI endonuclease or CRISPR, and a target locus is studied by targeted sequencing of PCR amplicons. “the median size of inserts is 120 bp [...] 39% of inserts originate from repetitive sequences, including telomeres, centromeres, Alu, and retrotransposon elements” “Inhibition [with NU7441] of DNA-PK catalytic activity [involved in NHEJ] significantly decreased insertion events. In contrast, the size and number of deletions increased.” “POLQ inhibition did not reduce [...] insertions, indicating that insertions were largely independent of microhomology-mediated end joining (MMEJ)” “Treatment with CK-666, a small-molecule inhibitor that stabilizes the Arp2/3 complex in an inactive conformation, resulted in a significant decrease in insertion events originating from AsiSI-proximal donor sequences” “siRNA-mediated downregulation of RAD51 did not affect the overall number of insertions but dramatically [increased] distal (class 3) insertions”. “8.7% of insertions originated from multiple genomic loci on different chromosomes” “more than 70% of donor sequences originated from promoters and transcribed gene body regions” “Donor sequences are spread around the AsiSI sites and span the TSS; however, insertions preferentially originate from the transcribed side [...]. In contrast, donor sequences originating from AsiSI-cleaved DSBs within a gene body or distal intergenic regions did not show any bias” ”[Insertions in] a Cas9 DSB near the TSS of the BCL6 locus in a [the OCI-Ly7 cell line] were markedly enriched in the direction of transcription.” “alpha-amanitin decreased the frequency of insertions, preferentially those originating from promoter-proximal DSBs [...]. Furthermore, donor sequences in alpha-amanitin-treated cells were no longer preferentially derived from the transcribed side.” “[Upregulating transcription of telomeric repeat-containing RNA at telomerse via] FANCM downregulation resulted in a significant increase in telomeric insertions.” “RNA-DNA hybrid disruption by RNaseH1 suppressed the bias of donor sequences that originate preferentially from the direction of transcription”