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.
We created waxy corn hybrids by CRISPR-Cas9 editing of a waxy allele in 12 elite inbred maize lines, a process that was more than a year faster than conventional trait introgression using ...backcrossing and marker-assisted selection. Field trials at 25 locations showed that CRISPR-waxy hybrids were agronomically superior to introgressed hybrids, producing on average 5.5 bushels per acre higher yield.
To expand the repertoire of Cas9s available for genome targeting, we present a new in vitro method for the simultaneous examination of guide RNA and protospacer adjacent motif (PAM) requirements. The ...method relies on the in vitro cleavage of plasmid libraries containing a randomized PAM as a function of Cas9-guide RNA complex concentration. Using this method, we accurately reproduce the canonical PAM preferences for Streptococcus pyogenes, Streptococcus thermophilus CRISPR3 (Sth3), and CRISPR1 (Sth1). Additionally, PAM and sgRNA solutions for a novel Cas9 protein from Brevibacillus laterosporus are provided by the assay and are demonstrated to support functional activity in vitro and in plants.
Retention of juvenile traits in the adult reproductive phase characterizes a process known as neoteny, and speculation exists over whether it has contributed to the evolution of new species. The ...dominant Corngrass1 (Cg1) mutant of maize is a neotenic mutation that results in phenotypes that may be present in the grass-like ancestors of maize. We cloned Cg1 and found that it encodes two tandem miR156 genes that are overexpressed in the meristem and lateral organs. Furthermore, a target of Cg1 is teosinte glume architecture1 (tga1), a gene known to have had a role in the domestication of maize from teosinte. Cg1 mutant plants overexpressing miR156 have lower levels of mir172, a microRNA that targets genes controlling juvenile development. By altering the relative levels of both microRNAs, it is possible to either prolong or shorten juvenile development in maize, thus providing a mechanism for how species-level heterochronic changes can occur in nature.
Development of anthers and pollen represents an important aspect of the life cycle in flowering plants. Genes contributing to anther and pollen development have been widely studied in many plant ...species. Ms26/CYP704B genes play an important role in pollen development through biosynthesis of sporopollenin for pollen exine formation. To investigate the role of Ms26/CYP704B genes in anther and pollen development of bread wheat, mutations in the A-, B-, and D-homeologs of the putative Ms26/CYP704B gene were analyzed. Single and double homozygous mutants in any of the homeologs did not affect pollen development and male fertility. Triple homozygous mutants resulted in completely male sterile plants that were defective in pollen and anther development. Additionally, double homozygous-single heterozygous mutants were also male sterile although with varying levels of residual fertility. The fertility of these triple mutants was dependent upon the homeolog contributing the wild-type allele. Two heterologous Ms26/CYP704B genes, when transformed into a triple homozygous mutant background, completely restored male fertility, whereas a single gene was unable to restore fertility. Functional analysis of Ms26/CYP704B furthers the understanding of male fertility genes which can be utilized for the development of novel hybrid seed production systems in wheat.
The current rate of yield gain in crops is insufficient to meet the predicted demands. Capturing the yield boost from heterosis is one of the few technologies that offers rapid gain. Hybrids are ...widely used for cereals, maize and rice, but it has been a challenge to develop a viable hybrid system for bread wheat due to the wheat genome complexity, which is both large and hexaploid. Wheat is our most widely grown crop providing 20% of the calories for humans. Here, we describe the identification of Ms1, a gene proposed for use in large-scale, low-cost production of male-sterile (ms) female lines necessary for hybrid wheat seed production. We show that Ms1 completely restores fertility to ms1d, and encodes a glycosylphosphatidylinositol-anchored lipid transfer protein, necessary for pollen exine development. This represents a key step towards developing a robust hybridization platform in wheat.Heterosis can rapidly boost yield in crop species but development of hybrid-breeding systems for bread wheat remains a challenge. Here, Tucker et al. describe the molecular identification of the wheat Ms1 gene and discuss its potential for large-scale hybrid seed production in wheat.
Hybrid varieties can provide the boost needed to increase stagnant wheat yields through heterosis. The lack of an efficient hybridization system, which can lower the cost of goods of hybrid seed ...production, has been a major impediment to commercialization of hybrid wheat varieties. In this review, we discuss the progress made in characterization of nuclear genetic male sterility (NGMS) in wheat and its advantages over two widely referenced hybridization systems, i.e., chemical hybridizing agents (CHAs) and cytoplasmic male sterility (CMS). We have characterized four wheat genes, i.e., Ms1, Ms5, TaMs26 and TaMs45, that sporophytically contribute to male fertility and yield recessive male sterility when mutated. While Ms1 and Ms5 are Triticeae specific genes, analysis of TaMs26 and TaMs45 demonstrated conservation of function across plant species. The main features of each of these genes is discussed with respect to the functional contribution of three sub-genomes and requirements for complementation of their respective mutants. Three seed production systems based on three genes, MS1, TaMS26 and TaMS45, were developed and a proof of concept was demonstrated for each system. The Tams26 and ms1 mutants were maintained through a TDNA cassette in a Seed Production Technology-like system, whereas Tams45 male sterility was maintained through creation of a telosome addition line. These genes represent different options for hybridization systems utilizing NGMS in wheat, which can potentially be utilized for commercial-scale hybrid seed production.
RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using ...Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.
Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants ...are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases.
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