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.

Arabidopsis mutants produced by constitutive overexpression of the CRISPR/Cas9 genome editing system are usually mosaics in the T1 generation. In this study, we used egg cell-specific promoters to drive the expression of Cas9 and obtained non-mosaic T1 mutants for multiple target genes with high efficiency. Comparisons of 12 combinations of eight promoters and two terminators found that the efficiency of the egg cell-specific promoter-controlled CRISPR/Cas9 system depended on the presence of a suitable terminator, and the composite promoter generated by fusing two egg cell-specific promoters resulted in much higher efficiency of mutation in the T1 generation compared with the single promoters.

Prime editing is a novel and universal CRISPR/Cas-derived precision genome-editing technology that has been recently developed. However, low efficiency of prime editing has been shown in transgenic rice lines. We hypothesize that enhancing pegRNA expression could improve prime-editing efficiency. In this report, we describe two strategies for enhancing pegRNA expression. We construct a prime editing vector harboring two pegRNA variants for W542L and S621I double mutations in ZmALS1 and ZmALS2. Compared with previous reports in rice, we achieve much higher prime-editing efficiency in maize. Our results are inspiring and provide a direction for the optimization of plant prime editors.

We present novel observations of high-specificity SpCas9 variants, sgRNA expression strategies based on mutant sgRNA scaffold and tRNA processing system, and CRISPR/Cas9-mediated T-DNA integrations.

Summary: Using genetic resistance against bacterial blight (BB) caused byXanthomonas oryzaepathovaroryzae(Xoo) is a major objective in rice breeding programmes. Prime editing (PE) has the potential to create novel germplasm againstXoo. Here, we use an improved prime‐editing system to implement two new strategies for BB resistance. Knock‐in of TAL effector binding elements (EBE) derived from the BB susceptible geneSWEET14into the promoter of a dysfunctional executorRgenexa23reaches 47.2% with desired edits including biallelic editing at 18% in T₀generation that enables an inducible TALE‐dependent BB resistance. Editing the transcription factor TFIIA geneTFIIAγ5required for TAL effector‐dependent BB susceptibility recapitulates the resistance ofxa5at an editing efficiency of 88.5% with biallelic editing rate of 30% in T₀generation. The engineered loci provided resistance against multipleXoostrains in T₁generation. Whole‐genome sequencing detected noOsMLH1dn‐associated random mutations and no off‐target editing demonstrating high specificity of this PE system. This is the first‐ever report to use PE system to engineer resistance against biotic stress and to demonstrate knock‐in of 30‐nucleotides cis‐regulatory element at high efficiency. The new strategies hold promises to fend rice off the evolvingXoostrains and protect it from epidemics.