A CRISPR/Cas9 toolkit for multiplex genome editing in plants

TLDR

The authors created a CRISPR/Cas9 toolkit that enables rapid, multiplex genome editing across diverse plant species by providing selectable markers, expanded gRNA modules, and simplified assembly. The toolkit consists of pGreen/pCAMBIA-based binary vectors and a BsaI‑compatible gRNA module set that allows maize‑codon‑optimized Cas9 and multiple gRNAs to be assembled in a single cloning step. Validation in maize protoplasts, transgenic maize, and Arabidopsis showed the system achieves high efficiency and specificity, generating heritable mutations in up to three genes in the T1 generation.

Abstract

To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required. We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation. We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.