A CRISPR-induced DNA break can trigger crossover, chromosomal loss and chromothripsis-like rearrangements

Abstract

The fate of DNA double-strand breaks (DSBs) generated by the Cas9 nuclease has been thoroughly studied. Repair via non-homologous end-joining (NHEJ) or homologous recombination (HR) is the common outcome. However, little is known about unrepaired DSBs and the type of damage they can trigger in plants. In this work, we designed a new assay that detects loss of heterozygosity (LOH) in somatic cells, enabling the study of a broad range of DSB-induced genomic events. The system relies on a mapped phenotypic marker which produces a light purple color (Betalain pigment) in all plant tissues. Plants with sectors lacking the Betalain marker upon DSB induction between the marker and the centromere were tested for LOH events. Using this assay we detected a flower with a twin yellow and dark purple sector, corresponding to a germinally transmitted somatic crossover event. We also identified instances of small deletions of genomic regions spanning the T-DNA and whole chromosome loss. In addition, we show that major chromosomal rearrangements including loss of large fragments, inversions, and translocations were clearly associated with the CRISPR-induced DSB. Detailed characterization of complex rearrangements by whole genome sequencing, molecular, and cytological analyses, supports a model in which breakage-fusion-bridge cycle followed by chromothripsis-like rearrangements had been induced. Our LOH assay provides a new tool for precise breeding via targeted crossover detection. It also uncovers CRISPR mediated chromothripsis-lke events that had not been previously identified in plants.

Plant phenotyping relevance

植物組織の色素表現型を利用して体細胞LOHや染色体イベントを検出する新規アッセイを開発しており、表現型に基づく測定法が研究の中心である。

Code and data availability

The paper describes plant phenotyping (Betalain sector screening, fasciation phenotypes) and WGS coverage analysis of tomato plants. The authors state that analysis code is public ('The R code used for plotting and testing of the rearrangements can be found at github.com/fabrimafe/CRISPRcoverage'), which is a paper-­‐­

No evidence-backed public reproduction asset is currently recorded.