Mutants in mitochondrial DNA repair genes in Arabidopsis thaliana generated by using CRISPR‐Cas9

Abstract

The genomes of plant mitochondria exhibit unique mutation rates and structural variability. Compared to their animal counterparts, plant mitochondria have low mutation rates, high recombination rates, and non‐circular genomes. Recent evidence suggests the prevalence of double‐strand break repair (DSBR) in plant mitochondria as one possible cause of these traits. Interestingly, all of the genes required for plant mitochondrial DSBR are located in the nucleus.In order to study DSBR, it is necessary to be able to reliably generate single and double‐mutants for DSBR genes. Using traditional methods, such as T‐DNA insertions, double mutant generation has proven cumbersome due to the occasional difficulty in PCR‐based identification of single mutants, as well as the low map distance between several genes of interest. CRISPR‐induced knockouts allow us to overcome some of these difficulties and generate a greater variety of mutants in a shorter period of time. In this study, we selected five proteins related to the processing of double strand breaks (DSBs) in plant mitochondria for mutagenesis: MSH1, RECA3, RNaseH1, SUV3, and OTP87. We prepared CRISPR Cas9 vectors to produce knockouts for each of these genes in Arabidopsis thaliana. The reliable production of mutants for these 5 genes will allow for in depth study of DSBR in the future. CRISPR Cas9 constructs were designed to produce two guide RNAs for each gene, targeting two different exons in the gene of interest, to give a better chance of successful knockouts, or deletions, between the two sites. These constructs were then transformed into Agrobacterium. Successful transformants were selected using a spectinomycin resistance gene in the construct. We then used a standard floral dip protocol to generate mutant Arabidopsis plants. PCR analysis confirmed Arabidopsis knockout mutations in all five lines.The mutants generated in this study will allow us to investigate several avenues of research on DSBR. We plan to expose plants with knockouts in either one or two DSBR genes to ciprofloxacin, an antibiotic which induces double strand breaks in plant mitochondrial DNA. From there we can observe the mitochondrial genome recombination rates using PCR and Illumina sequencing for each of the mutants.Support or Funding InformationThis work was supported in part by grants from the National Science Foundation to A.C.C. (MCB‐1413152 and MCB‐1933590) and a grant from the UNL UCARE program (26‐0101‐8001‐001)