A Fast and Cost-Effective Genotyping Method for CRISPR-Cas9-Generated Mutant Rice Lines

Abstract

With increasing throughput in both the generation and phenotyping of mutant lines in plants, it is important to have an efficient and reliable genotyping method. Traditional workflows, still commonly used in many labs, have time-consuming and expensive steps, such as DNA purification, cloning and growing E. coli cultures. We propose an alternative workflow where these steps are bypassed, using Phire polymerase on fresh plant tissue, and ExoProStar treatment as preparation for sequencing. We generated CRISPR-Cas9 mutants for ZAS (ZAXINONE SYNTHASE) in rice with two guide RNAs. Using both a traditional workflow and our proposed workflow, we genotyped nine T1 plants. To interpret the sequencing output, which is often complex in CRISPR-generated mutants, we used free online automatic analysis systems and compared the results. Our proposed workflow produces results of the same quality as the old workflow, but in 1 day instead of 3 days and about 35 times cheaper. This workflow also consists of fewer steps and reduces the risk of cross contamination and mistakes. Furthermore, the automated sequence analysis packages are mostly accurate and could easily be used for bulk analysis. Based on these advantages, we encourage academic and commercial labs conducting genotyping to consider switching over to our proposed workflow.

Keywords

• CRISPR-Cas9
• genotyping
• PCR-direct
• transgenicity
• sanger sequencing
• ZAXINONE SYNTHASE