RNA-guided AsCas12a- and SpCas9-catalyzed knockout and homology directed repair of the omega-1 locus of the human blood fluke, Schistosoma mansoni

We compared the efficiency and precision of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. with SpCas9 of Streptococcus pyogenes aiming to advance functional genomics tools for Schistosoma mansoni. Programmed double stranded cleavage catalyzed by AsCas12a results in a staggered strand break whereas SpCas9 produces a blunt ended chromosomal break. The TTTV, the optimal protospacer adjacent motif for AsCas12a is expected frequently within the AT-rich genome of this platyhelminth. We deployed optimized conditions (gRNA:SpCas9:DNA donor ratio and electroporation condition) to edit the ω1 gene. SpCas9 delivered higher efficiency to mutate ω1 target compared to AsCas12a for non-homology end joining (NHEJ)-catalyzed repair (14.04% vs. 10.88%, n = 7 replicates). Most mutations were deletions; SpCas9 induced -3 nt size deletions whereas AsCas12a induced deletions ranging in size from -25 to -2 nt. Although these were less absolute percentage AsCas12a than SpCas9 programmed mutations, the phenotypic outcomes on levels of ω1 transcripts and expressed omega-1 protein were similar. Gene editing efficiency of SpCas9 and AsCas12a markedly increased in the presence of short single stranded donor templates bearing symmetrical homolog arms of 50 nt in length. With AsCas12a, both non-CRISPR target (NT) and target (T) strands of the ω1 gene were tested as homology direct repair (HDR) donor templates. There were 15.67%, 28.71% and 21.43% of NHEJ from 7 pooled genomic DNA from KI_SpCas9, KI_AsCas12a-NT-ssODN and KI_AsCas12a-T-ssODN experiments, respectively. Programmed SpCas9 cleavage led to higher levels than AsCas12a of precise HDR mediated; 17.07%, KI_SpCas9 vs. 14.58%, KI_AsCas12a-NT-ssODN and 12.35%, KI_AsCas12a-T-ssODN (P < 0.0.5), although no significant differences in reduction in ω1 transcripts or of protein levels were apparent. These findings revealed that both AsCas12a and SpCas9 can provide programmed knockout and transgene insertion into genes expressed in the schistosome egg.

High Temperature Drives Topoisomerase Mediated Chromosomal Break Repair Pathway Choice

Cancer-causing mutations often arise from inappropriate DNA repair, yet acute exposure to DNA damage is widely used to treat cancer. The challenge remains in how to specifically induce excessive DNA damage in cancer cells while minimizing the undesirable effects of genomic instability in noncancerous cells. One approach is the acute exposure to hyperthermia, which suppresses DNA repair and synergizes with radiotherapy and chemotherapy. An exception, however, is the protective effect of hyperthermia on topoisomerase targeting therapeutics. The molecular explanation for this conundrum remains unclear. Here, we show that hyperthermia suppresses the level of topoisomerase mediated single- and double-strand breaks induced by exposure to topoisomerase poisons. We further uncover that, hyperthermia suppresses hallmarks of genomic instability induced by topoisomerase targeting therapeutics by inhibiting nuclease activities, thereby channeling repair to error-free pathways driven by tyrosyl-DNA phosphodiesterases. These findings provide an explanation for the protective effect of hyperthermia from topoisomerase-induced DNA damage and may help to explain the inverse relationship between cancer incidence and temperature. They also pave the way for the use of controlled heat as a therapeutic adjunct to topoisomerase targeting therapeutics.

PML isoform expression and DNA break location relative to PML nuclear bodies impacts the efficiency of homologous recombination

Promyelocytic leukemia nuclear bodies (PML NBs) are nuclear subdomains that respond to genotoxic stress by increasing in number via changes in chromatin structure. However, the role of the PML protein and PML NBs in specific mechanisms of DNA repair has not been fully characterized. Here, we have directly examined the role of PML in homologous recombination (HR) using I-SceI extrachromosomal and chromosome-based homology-directed repair (HDR) assays, and in HDR by CRISPR/Cas9-mediated gene editing. We determined that PML loss can inhibit HR in an extrachromosomal HDR assay but had less of an effect on CRISPR/Cas9-mediated chromosomal HDR. Overexpression of PML also inhibited both CRISPR HDR and I-SceI-induced HDR using a chromosomal reporter, and in an isoform-specific manner. However, the impact of PML overexpression on the chromosomal HDR reporter was dependent on the intranuclear chromosomal positioning of the reporter. Specifically, HDR at the TAP1 gene locus, which is associated with PML NBs, was reduced compared with a locus not associated with a PML NB; yet, HDR could be reduced at the non-PML NB-associated locus by PML overexpression. Thus, both loss and overexpression of PML isoforms can inhibit HDR, and proximity of a chromosomal break to a PML NB can impact HDR efficiency.

RNF8 has both KU-dependent and independent roles in chromosomal break repair

Chromosomal double strand breaks (DSBs) can initiate several signaling events, such as ubiquitination, however the precise influence of such signaling on DSB repair outcomes remains poorly understood. With an RNA interference screen, we found that the E3 ubiquitin ligase RNF8 suppresses a deletion rearrangement mediated by canonical non-homologous end joining (C-NHEJ). We also found that RNF8 suppresses EJ without insertion/deletion mutations, which is a hallmark of C-NHEJ. Conversely, RNF8 promotes alternative EJ (ALT-EJ) events involving microhomology that is embedded from the edge of the DSB. These ALT-EJ events likely require limited end resection, whereas RNF8 is not required for single-strand annealing repair involving extensive end resection. Thus, RNF8 appears to specifically facilitate repair events requiring limited end resection, which we find is dependent on the DSB end protection factor KU. However, we also find that RNF8 is important for homology-directed repair (HDR) independently of KU, which appears linked to promoting PALB2 function. Finally, the influence of RNF8 on EJ is distinct from 53BP1 and the ALT-EJ factor, POLQ. We suggest that RNF8 mediates both ALT-EJ and HDR, but via distinct mechanisms, since only the former is dependent on KU.

Tetratricopeptide repeat factor XAB2 mediates the end resection step of homologous recombination

We examined the influence of the tetratricopeptide repeat factor XAB2 on chromosomal break repair, and found that XAB2 promotes end resection that generates the 3′ ssDNA intermediate for homologous recombination (HR). Namely, XAB2 is important for chromosomal double-strand break (DSB) repair via two pathways of HR that require end resection as an intermediate step, end resection of camptothecin (Cpt)-induced DNA damage, and RAD51 recruitment to ionizing radiation induced foci (IRIF), which requires end resection. Furthermore, XAB2 mediates specific aspects of the DNA damage response associated with end resection proficiency: CtIP hyperphosphorylation induced by Cpt and BRCA1 IRIF. XAB2 also promotes histone acetylation events linked to HR proficiency. From truncation mutation analysis, the capacity for XAB2 to promote HR correlates with its ability to form a complex with ISY1 and PRP19, which show a similar influence as XAB2 on HR. This XAB2 complex localizes to punctate structures consistent with interchromatin granules that show a striking adjacent-localization to the DSB marker γH2AX. In summary, we suggest that the XAB2 complex mediates DNA damage response events important for the end resection step of HR, and speculate that its adjacent-localization relative to DSBs marked by γH2AX is important for this function.