CRISPR targeting of MEIOTIC-TOPOISOMERASE VIB-dCas9 to a recombination hotspot is insufficient to increase crossover frequency in Arabidopsis

Abstract

During meiosis, homologous chromosomes pair and recombine, which can result in reciprocal crossovers that increase genetic diversity. Crossovers are unevenly distributed along eukaryote chromosomes and show repression in heterochromatin and the centromeres. Within the chromosome arms crossovers are often concentrated in hotspots, which are typically in the kilobase range. The uneven distribution of crossovers along chromosomes, together with their low number per meiosis, creates a limitation during crop breeding, where recombination can be beneficial. Therefore, targeting crossovers to specific genome locations has the potential to accelerate crop improvement. In plants, meiotic crossovers are initiated by DNA double strand breaks (DSBs) that are catalysed by SPO11 complexes, which consist of two catalytic (SPO11-1 and SPO11-2) and two non-catalytic subunits (MTOPVIB). We used the model plant Arabidopsis thaliana to target a dCas9-MTOPVIB fusion protein to the 3a crossover hotspot via CRISPR. We observed that this was insufficient to significantly change meiotic crossover frequency or pattern within 3a. We discuss the implications of our findings for targeting meiotic recombination within plant genomes.

Rewiring Meiosis for Crop Improvement

“…Studies from budding yeast revealed that not every locus bound by SPO11-GAL4 is proficient to form DSBs, and the establishment of a DSB is determined by local factors ( Robine et al, 2007 ; Sarno et al, 2017 ). In plants, a recent study suggests that expression of a MTOPVIB-dCas9 fusion protein to induce targeted meiotic DSB within a CO hotspot located in a subtelomeric region of chromosome 3 is not sufficient to affect CO frequency ( Yelina et al, 2021 ). In budding yeast, it is estimated that around 40% of the DSBs are converted to COs ( Mancera et al, 2008 ).…”

Manipulation of Meiotic Recombination to Hasten Crop Improvement

“…In plants, a dCas9::mTOPVIB fusion protein has been accumulated under the control of the mTopVIB promoter in a null mtopVIB mutant of Arabidopsis for targeting DSB at a CO HS locus via the simultaneous expression of 6 sgRNAs. However, this did not significantly enhance the local meiotic CO frequency, possibly because the frequency was naturally high at this locus [85]. The interactions existing between the members of the catalytic subcomplex (AtSPO11-1, AtSPO11-2, mTOPVIB) with the other DSB proteins, belonging either to the RMM-like complex (namely DFO, PRD2/AtMEI4, pHS1/Rec114), ensuring the anchoring of the catalytic complex to the axis, or to the DSB resection complex (COM1, RAD50, MRE11, and NBS1) through an interaction with PRD1 itself interacting with PRD3/MER2, have been recently clarified in Arabidopsis [86].…”

Fast and Precise: How to Measure Meiotic Crossovers in Arabidopsis

“…At a single CO hotspot, DNA methylation or arp6 mutation was shown to induce fewer COs ( Choi et al, 2013 ; Yelina et al, 2015 ). As promoting COs in specific regions is of great interest in plant breeding, the CRISPR/dCas9 targeting approach was applied to increase CO frequency at a single CO hotspot in Arabidopsis using MTOPVIB (meiotic topoisomerase VIB)-dCas9 fusion, but the moderate effect reflected the complexity of meiotic recombination control ( Yelina et al, 2021 ). Two MTOPVIB proteins interact with SPO11-1 and SPO11-2 topoisomerase-like proteins that catalyze meiotic DSB formation.…”