Transformation in grass species has traditionally relied on immature embryos and has therefore been limited to a few major Poaceae crops. Other transformation explants, including leaf tissue, have been explored but with low success rates, which is one of the major factors hindering the broad application of genome editing for crop improvement. Recently, leaf transformation using morphogenic genes Wuschel2 (Wus2) and Babyboom (Bbm) has been successfully used for Cas9-mediated mutagenesis, but complex genome editing applications, requiring large numbers of regenerated plants to be screened, remain elusive. Here we demonstrate that enhanced Wus2/Bbm expression substantially improves leaf transformation in maize and sorghum, allowing the recovery of plants with Cas9-mediated gene dropouts and targeted gene insertion. Moreover, using a maize-optimized Wus2/Bbm construct, embryogenic callus and regenerated plantlets were successfully produced in eight species spanning four grass subfamilies, suggesting that this may lead to a universal family-wide method for transformation and genome editing across the Poaceae.

SummaryCRISPR‐Cas is a powerful DNA double‐strand break technology with wide‐ranging applications in plant genome modification. However, the efficiency of genome editing depends on various factors including plant genetic transformation processes and types of modifications desired. Agrobacterium infection is the preferred method of transformation and delivery of editing components into the plant cell. While this method has been successfully used to generate gene knockouts in multiple crops, precise nucleotide replacement and especially gene insertion into a pre‐defined genomic location remain highly challenging. Here, we report an efficient, selectable marker‐free site‐specific gene insertion in maize using Agrobacterium infection. Advancements in maize transformation and new vector design enabled increase of targeted insertion frequencies by two orders of magnitude in comparison to conventional Agrobacterium‐mediated delivery. Importantly, these advancements allowed not only a significant improvement of the frequency, but also of the quality of generated events. These results further enable the application of genome editing for trait product development in a wide variety of crop species amenable to Agrobacterium‐mediated transformation.

Despite the conspicuous and rapid development of genome editing tools, implementing this technology in plants often remains constrained by our inability to regenerate fertile genome-modified plants. Finding a viable combination of delivery method, genome editing reagents, and plant regeneration system has often been both crop-specific and crop-limited. Recent progress using morphogenic genes such as Wuschel2 (Wus2) and Babyboom (Bbm) has alleviated some of these crop-specific challenges, and along with other improvements, continues to expand the range of plants that can be edited.

CRISPR–Cas is a powerful double-strand-break technology with wide-ranging applications from gene discovery to commercial product development. Thus far, this tool has been almost exclusively used for gene knockouts and deletions, with a few examples of gene edits and targeted gene insertions. Here, we demonstrate the application of CRISPR–Cas9 technology to mediate targeted 75.5-Mb pericentric inversion in chromosome 2 in one of the elite maize inbred lines from Corteva Agriscience. This inversion unlocks a large chromosomal region containing substantial genetic variance for recombination, thus providing opportunities for the development of new maize varieties with improved phenotypes.

CRISPR-Cas9 is a powerful tool for generating targeted mutations and genomic deletions. However, precise gene insertion or sequence replacement remains a major hurdle before application of CRISPR-Cas9 technology is fully realized in plant breeding. Here, we report high-frequency, selectable marker-free intra-genomic gene targeting (GT) in maize. Heat shock-inducible Cas9 was used for generating targeted double-strand breaks and simultaneous mobilization of the donor template from pre-integrated T-DNA. The construct was designed such that release of the donor template and subsequent DNA repair activated expression of the selectable marker gene within the donor locus. This approach generated up to 4.7% targeted insertion of the donor sequence into the target locus in T0 plants, with up to 86% detected donor template release and 99% mutation rate being observed at the donor loci and the genomic target site, respectively. Unlike previous in planta or intra-genomic homologous recombination reports in which the original chimeric GT plants required extensive progeny screening in the next generation to identify non-chimeric GT individuals, our method provides non-chimeric heritable GT in one generation.

CRISPR-Cas is a powerful DNA double strand break technology with wide-ranging applications in plant genome modification. However, the efficiency of genome editing depends on various factors including plant genetic transformation processes and types of modifications desired. Agrobacterium infection is the preferred method of transformation and delivery of editing components into the plant cell. While this method has been successfully used to generate gene knock-outs in multiple crops, precise nucleotide replacement and especially gene insertion into a pre-defined genomic location remain highly challenging. Here we report an efficient, heritable, selectable marker-free site-specific gene insertion in maize using Agrobacterium -mediated delivery. Advancements in maize transformation and new vector design enabled targeted insertion with frequencies as high as 8–10%. Importantly, these advancements allowed not only an improvement of the frequency but also of the quality of generated events. These results further enable the application of genome editing for trait product development in a wide variety of crop species amenable to Agrobacterium -mediated transformation.

Synthetic biology is still a nascent area in plant biology. Synthetic biology can be defined simply as applying engineering principles to biology. It relies on designing, building, and testing components, just like engineers do, when making a device. In our case the device is a modified plant consisting of new ‘parts.’The parts are mostly made of DNA. They are genes, gene expression regulators, parts of genes, or, collections of genes. So far, the biggest application of synthetic biology in plants is gene editing using CRISPR systems–essentially ‘rewriting’ genes– but my goal is to extend the full power of synthetic biology into ‘writing’ DNA at a large scale into genomes. Theoretically the ‘writing’ can result in the entire genome of plants, which is far beyond what is currently feasible. The best I’ve hoped for is to rewrite the chloroplast genome of 100 genes: a synthetic chloroplast genome or “synplastome.” I will discuss the work of my group toward designing, building and installing the first synplastome in plants as well as mini-synplastomes.

CRISPR-Cas9 enabled genome engineering has great potential for improving agriculture productivity, but the possibility of unintended off-target edits has evoked some concerns. Here we employ a three-step strategy to investigate Cas9 nuclease specificity in a complex plant genome. Our approach pairs computational prediction with genome-wide biochemical off-target detection followed by validation in maize plants. Our results reveal high frequency (up to 90%) on-target editing with no evidence of off-target cleavage activity when guide RNAs were bioinformatically predicted to be specific. Predictable off-target edits were observed but only with a promiscuous guide RNA intentionally designed to validate our approach. Off-target editing can be minimized by designing guide RNAs that are different from other genomic locations by at least three mismatches in combination with at least one mismatch occurring in the PAM proximal region. With well-designed guides, genetic variation from Cas9 off-target cleavage in plants is negligible, and much less than inherent variation.

Summary Targeted mutagenesis using programmable DNA endonucleases has broad applications for studying gene function in planta and developing approaches to improve crop yields. Recently, a genetic method that eliminates the need to emasculate the female inbred during hybrid seed production, referred to as Seed Production Technology, has been described. The foundation of this genetic system relied on classical methods to identify genes critical to anther and pollen development. One of these genes is a P450 gene which is expressed in the tapetum of anthers. Homozygous recessive mutants in this gene render maize and rice plants male sterile. While this P450 in maize corresponds to the male fertility gene Ms26 , male fertility mutants have not been isolated in other monocots such as sorghum and wheat. In this report, a custom designed homing endonuclease, Ems26+, was used to generate in planta mutations in the rice, sorghum and wheat orthologs of maize Ms26 . Similar to maize, homozygous mutations in this P450 gene in rice and sorghum prevent pollen formation resulting in male sterile plants and fertility was restored in sorghum using a transformed copy of maize Ms26 . In contrast, allohexaploid wheat plants that carry similar homozygous nuclear mutations in only one, but not all three, of their single genomes were male fertile. Targeted mutagenesis and subsequent characterization of male fertility genes in sorghum and wheat is an important step for capturing heterosis and improving crop yields through hybrid seed.

Targeted DNA double-strand breaks have been shown to significantly increase the frequency and precision of genome editing. In the past two decades, several double-strand break technologies have been developed. CRISPR–Cas9 has quickly become the technology of choice for genome editing due to its simplicity, efficiency and versatility. Currently, genome editing in plants primarily relies on delivering double-strand break reagents in the form of DNA vectors. Here we report biolistic delivery of pre-assembled Cas9–gRNA ribonucleoproteins into maize embryo cells and regeneration of plants with both mutated and edited alleles. Using this method of delivery, we also demonstrate DNA- and selectable marker-free gene mutagenesis in maize and recovery of plants with mutated alleles at high frequencies. These results open new opportunities to accelerate breeding practices in a wide variety of crop species.

Targeted mutagenesis, editing of endogenous maize (Zea mays) genes, and site-specific insertion of a trait gene using clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)-guide RNA technology are reported in maize. DNA vectors expressing maize codon-optimized Streptococcus pyogenes Cas9 endonuclease and single guide RNAs were cointroduced with or without DNA repair templates into maize immature embryos by biolistic transformation targeting five different genomic regions: upstream of the liguleless1 (LIG1) gene, male fertility genes (Ms26 and Ms45), and acetolactate synthase (ALS) genes (ALS1 and ALS2). Mutations were subsequently identified at all sites targeted, and plants containing biallelic multiplex mutations at LIG1, Ms26, and Ms45 were recovered. Biolistic delivery of guide RNAs (as RNA molecules) directly into immature embryo cells containing preintegrated Cas9 also resulted in targeted mutations. Editing the ALS2 gene using either single-stranded oligonucleotides or double-stranded DNA vectors as repair templates yielded chlorsulfuron-resistant plants. Double-strand breaks generated by RNA-guided Cas9 endonuclease also stimulated insertion of a trait gene at a site near LIG1 by homology-directed repair. Progeny showed expected Mendelian segregation of mutations, edits, and targeted gene insertions. The examples reported in this study demonstrate the utility of Cas9-guide RNA technology as a plant genome editing tool to enhance plant breeding and crop research needed to meet growing agriculture demands of the future.

Targeted mutagenesis, editing of endogenous maize (Zea mays) genes, and site-specific insertion of a trait gene using clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)-guide RNA technology are reported in maize. DNA vectors expressing maize codon-optimized Streptococcus pyogenes Cas9 endonuclease and single guide RNAs were cointroduced with or without DNA repair templates into maize immature embryos by biolistic transformation targeting five different genomic regions: upstream of the liguleless1 (LIG1) gene, male fertility genes (Ms26 and Ms45), and acetolactate synthase (ALS) genes (ALS1 and ALS2). Mutations were subsequently identified at all sites targeted, and plants containing biallelic multiplex mutations at LIG1, Ms26, and Ms45 were recovered. Biolistic delivery of guide RNAs (as RNA molecules) directly into immature embryo cells containing preintegrated Cas9 also resulted in targeted mutations. Editing the ALS2 gene using either single-stranded oligonucleotides or double-stranded DNA vectors as repair templates yielded chlorsulfuron-resistant plants. Double-strand breaks generated by RNA-guided Cas9 endonuclease also stimulated insertion of a trait gene at a site near LIG1 by homology-directed repair. Progeny showed expected Mendelian segregation of mutations, edits, and targeted gene insertions. The examples reported in this study demonstrate the utility of Cas9-guide RNA technology as a plant genome editing tool to enhance plant breeding and crop research needed to meet growing agriculture demands of the future.