The CRISPR/Cas9 technology revolutionizes targeted gene knockout in diverse organisms including plants. However, screening edited alleles, particularly those with multiplex editing, from herbicide- or antibiotic-resistant transgenic plants and segregating out the Cas9 transgene represent two laborious processes. Current solutions to facilitate these processes rely on different selection markers. Here, by taking advantage of the opposite functions of a d-amino acid oxidase (DAO) in detoxifying d-serine and in metabolizing non-toxic d-valine to a cytotoxic product, we develop a DAO-based selection system that simultaneously enables the enrichment of multigene edited alleles and elimination of Cas9-containing progeny in Arabidopsis thaliana. Among five DAOs tested in Escherichia coli, the one encoded by Trigonopsis variabilis (TvDAO) could confer slightly stronger d-serine resistance than other homologs. Transgenic expression of TvDAO in Arabidopsis allowed a clear distinction between transgenic and non-transgenic plants in both d-serine-conditioned positive selection and d-valine-conditioned negative selection. As a proof of concept, we combined CRISPR-induced single-strand annealing repair of a dead TvDAO with d-serine-based positive selection to help identify transgenic plants with multiplex editing, where d-serine-resistant plants exhibited considerably higher co-editing frequencies at three endogenous target genes than those selected by hygromycin. Subsequently, d-valine-based negative selection successfully removed Cas9 and TvDAO transgenes from the survival offspring carrying inherited mutations. Collectively, this work provides a novel strategy to ease CRISPR mutant identification and Cas9 transgene elimination using a single selection marker, which promises more efficient and simplified multiplex CRISPR editing in plants.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s42994-023-00132-6.

The CRISPR/Cas9 genome-editing system has emerged as a popular powerful tool for biological research. However, the process of selecting efficiently edited Cas9-free plants is usually laborious and time consuming. Here, we demonstrated P2A to be the most efficient self-cleaving peptide for fusing Cas9 and GFP in Arabidopsis and then used Cas9-P2A-GFP to develop a novel CRISPR/Cas9 system. Additionally, a pair of isocaudomer restriction enzymes were selected to conveniently assemble multiple sgRNAs. In this system, the GFP fluorescence intensity in T1 transgenic plants indicates the expression level of the Cas9 protein, which correlates well with the editing efficiency. Furthermore, Cas9-free plants can be easily selected by examining GFP fluorescence in T2 transgenic plants. The efficient knockout of BRI1, BZR1 and BES1 demonstrated the robustness of our new system. Thus, we designed a novel CRISPR/Cas9 system that can generate Cas9-free multiplex mutants efficiently in Arabidopsis and possibly in other plant species.

We determined the specificity of mutations induced by the CRISPR-Cas9 gene-editing system in tobacco (Nicotiana benthamiana) alleles and subsequent genetic stability. For this, we prepared 248 mutant plants using an Agrobacterium-delivered CRISPR-Cas9 system targeting α-1,3-fucosyltransferase 1 (FucT1) and β-1,2-xylosyltransferase1 (XylT1) genes, for which the mutation rates were 22.5% and 25%, respectively, with 20.5% for both loci. Individuals with wild-type (WT) alleles at the NbFucT1 locus in T0 were further segregated into chimeric progeny (37-54%) in the next generation, whereas homozygous T0 mutants tended to produce more (~70%) homozygotes than other bi-allelic and chimeric progenies in the T1 generation. Approximately 81.8% and 77.4% of the homozygous and bi-allelic mutations in T0 generation, respectively, were stably inherited in the next generation, and approximately 50% of the Cas9-free mutants were segregated in T2 generation. One homozygous mutant (Ta 161-1) with a +1 bp insertion in NbFucT1 and a -4 bp deletion in NbXylT1 was found to produce T2 progenies with the same alleles, indicating no activity of the integrated Cas9 irrespective of the insertion or deletion type. Our results provide empirical evidence regarding the genetic inheritance of alleles at CRISPR-targeted loci in tobacco transformants and indicate the potential factors contributing to further mutagenesis.

CRISPR/Cas9 system directed by a gene-specific single guide RNA (sgRNA) is an effective tool for genome editing such as deletions of few bases in coding genes. However, targeted deletion of larger regions generate loss-of-function alleles that offer a straightforward starting point for functional dissections of genomic loci. We present an easy-to-use strategy including a fast cloning dual-sgRNA vector linked to efficient isolation of heritable Cas9-free genomic deletions to rapidly and cost-effectively generate a targeted heritable genome deletion. This step-by-step protocol includes gRNA design, cloning strategy and mutation detection for Arabidopsis and may be adapted for other plant species.