A screening method to identify efficient sgRNAs in Arabidopsis, used in conjunction with cell-specific lignin reduction.

Background: Single guide RNA (sgRNA) selection is important for the efficiency of CRISPR/Cas9-mediated genome editing. However, in plants, the rules governing selection are not well established.

Results: We developed a facile transient assay to screen sgRNA efficiency. We then used it to test top-performing bioinformatically predicted sgRNAs for two different Arabidopsis genes. In our assay, these sgRNAs had vastly different editing efficiencies, and these efficiencies were replicated in stably transformed Arabidopsis lines. One of the genes, -CoA (), is an essential gene, required for lignin biosynthesis. Previously, HCT function has been studied using gene silencing. Here, to avoid the negative growth effects that are due to the loss of HCT activity in xylem vessels, we used a fiber-specific promoter to drive CAS9 expression. Two independent transgenic lines showed the expected lignin decrease. Successful editing was confirmed via the observation of mutations at the target loci, as well as an approximately 90% decrease in HCT activity. Histochemical analysis and a normal growth phenotype support the fiber-specific knockout of . For the targeting of the second gene, - (), a highly efficient sgRNA drastically increased the rate of germline editing in T1 generation.

Conclusions: This screening method is widely applicable, and the selection and use of efficient sgRNAs will accelerate the process of expanding germplasm for both molecular breeding and research. In addition, this, to the best of our knowledge, is the first application of constrained genome editing to obtain chimeric plants of essential genes, thereby providing a dominant method to avoid lethal growth phenotypes.