Pollen self-elimination CRISPR-Cas genome editing prevents transgenic pollen dispersal in maize.

This study reports the development of a programmed pollen self-elimination CRISPR-Cas (PSEC) system in which the pollen is infertile when PSEC is present in haploid pollen. PSEC can be inherited through the female gametophyte and retains genome editing activity in vivo across generations. This system could greatly alleviate serious concerns about the widespread diffusion of genetically modified (GM) elements into natural and agricultural environments via outcrossing.

A straight-forward seed production technology system for foxtail millet (Setaria italica).

For autogamous crops, a precondition for using heterosis is to produce sufficient pure male-sterile female parents that can be used to produce hybrid seeds. To date, cytoplasmic male sterility (CMS) and environment-sensitive genic male sterility (EGMS) have been used commercially to exploit heterosis for autogamous species. However, neither CMS nor EGMS has been established for foxtail millet (Setaria italica). Here, we report on the establishment and application of a seed production technology (SPT) system for this crop. First, we established a DsRed-based SPT system, but found that it was unsuitable because it required the use of a fluorescent device for seed sorting. Instead, we constructed an SPT system with de novo betalain biosynthesis as the selection marker. This allowed us to distinguish transgenic seeds with the naked eye, thereby facilitating the identification of SPT maintainer line seeds. In this system, a seed sorter was not required to obtain sufficient seeds. The key point of the strategy is that the seed pool of the SPT maintainer line is propagated by artificial identification and harvesting of male-fertile individuals in the field, and the male-sterile line seed pool for hybrid production is produced and propagated by free pollination of male-sterile plants with the SPT maintainer line. In a field experiment, we obtained 423.96 kg male-sterile line seeds per acre, which is sufficient to plant 700.18 acres of farmland for hybrid seed production or male-sterile line reproduction. Our study therefore describes a powerful tool for hybrid seed production in foxtail millet, and demonstrates how the SPT system can be used for a small-grained crop with high reproduction efficiency.

CRISPR/dCas-mediated gene activation toolkit development and its application for parthenogenesis induction in maize.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems can be engineered as programmable transcription factors to either activate (CRISPRa) or inhibit transcription. Apomixis is extremely valuable for the seed industry in breeding clonal seeds with pure genetic backgrounds. We report here a CRISPR/dCas9-based toolkit equipped with dCas9-VP64 and MS2-p65-HSF1 effectors that may specifically target genes with high activation capability. We explored the application of in vivo CRISPRa targeting of maize BABY BOOM2 (ZmBBM2), acting as a fertilization checkpoint, as a means to engineer parthenogenesis. We detected ZmBBM2 transcripts only in egg cells but not in other maternal gametic cells. Activation of ZmBBM2 in egg cells in vivo caused maternal cell-autonomous parthenogenesis to produce haploid seeds. Our work provides a highly specific gene-activation CRISPRa technology for target cells and verifies its application for parthenogenesis induction in maize.

A field-deployable method for single and multiplex detection of DNA or RNA from pathogens using Cas12 and Cas13.

For some Cas nucleases, trans-cleavage activity triggered by CRISPR/Cas-mediated cis-cleavage upon target nucleic acid recognition has been explored for diagnostic detection. Portable single and multiplex nucleic acid-based detection is needed for crop pathogen management in agriculture. Here, we harnessed and characterized RfxCas13d as an additional CRISPR/Cas nucleic acid detection tool. We systematically characterized AsCas12a, LbCas12a, LwaCas13a, and RfxCas13d combined with isothermal amplification to develop a CRISPR/Cas nucleic acid-based tool for single or multiplex pathogen detection. Our data indicated that sufficient detection sensitivity was achieved with just a few copies of DNA/RNA targets as input. Using this tool, we successfully detected DNA from Fusarium graminearum and Fusarium verticillioides and RNA from rice black-streaked dwarf virus in crude extracts prepared in the field. Our method, from sample preparation to result readout, could be rapidly and easily deployed in the field. This system could be extended to other crop pathogens, including those that currently lack a detection method and have metabolite profiles that make detection challenging. This nucleic acid detection system could also be used for single-nucleotide polymorphism genotyping, transgene detection, and qualitative detection of gene expression in the field.

Establishment of an efficient seed fluorescence reporter-assisted CRISPR/Cas9 gene editing in maize.

Genome editing by clustered regularly interspaced short palindromic sequences (CRISPR)/CRISPR-associated protein 9 (Cas9) has revolutionized functional gene analysis and genetic improvement. While reporter-assisted CRISPR/Cas systems can greatly facilitate the selection of genome-edited plants produced via stable transformation, this approach has not been well established in seed crops. Here, we established the seed fluorescence reporter (SFR)-assisted CRISPR/Cas9 systems in maize (Zea mays L.), using the red fluorescent DsRED protein expressed in the endosperm (En-SFR/Cas9), embryos (Em-SFR/Cas9), or both tissues (Em/En-SFR/Cas9). All three SFRs showed distinct fluorescent patterns in the seed endosperm and embryo that allowed the selection of seeds carrying the transgene of having segregated the transgene out. We describe several case studies of the implementation of En-SFR/Cas9, Em-SFR/Cas9, and Em/En- SFR/Cas9 to identify plants not harboring the genome-editing cassette but carrying the desired mutations at target genes in single genes or in small-scale mutant libraries, and report on the successful generation of single-target mutants and/or mutant libraries with En-SFR/Cas9, Em-SFR/Cas9, and Em/En-SFR/Cas9. SFR-assisted genome editing may have particular value for application scenarios with a low transformation frequency and may be extended to other important monocot seed crops.

Genome Editing Enables Next-Generation Hybrid Seed Production Technology.

The next-generation hybrid seed technology enables the successful production of sortable hybrid seeds from genic male sterile (GMS) lines and maintainers; however, it requires multiple laborious and complicated steps. Here, we designed a simple next-generation hybrid seed production strategy that takes advantage of the CRISPR/Cas9 technology to create a Manipulated GMS Maintainer (MGM) system via a single transformation. Under this schema, the maize male fertility gene ZmMS26 was nullified by removal of its fifth exon using the CRISPR/Cas9 system on a vector, and a second vector carrying a functional ZmMS26 cDNA was co-transformed to restore fertility. The second vector also contains a male gametophyte inactivation gene (ZmAA1) encoding maize α-amylase driven by the pollen-specific promoter PG47 and an endosperm fluorescent marker (DsRED) driven by the barley endosperm aleurone-specific promoter Ltp2. The derived single-copy hemizygous MGM lines bore a mutated MS26 gene, leading to complete male sterility but normal vegetative growth and grain yield. The MGM system could prevent genetic transmission of the MGM elements via male gametophytes, providing an efficient method for sorting maintainer seeds labeled by DsRED. This strategy can be extended to any GMS gene and to hybrid crops other than maize.

RNA-guided Cas9 as an in vivo desired-target mutator in maize.

The RNA-guided Cas9 system is a versatile tool for genome editing. Here, we established a RNA-guided endonuclease (RGEN) system as an in vivo desired-target mutator (DTM) in maize to reduce the linkage drag during breeding procedure, using the LIGULELESS1 (LG1) locus as a proof-of-concept. Our system showed 51.5%-91.2% mutation frequency in T0 transgenic plants. We then crossed the T1 plants stably expressing DTM with six diverse recipient maize lines and found that 11.79%-28.71% of the plants tested were mutants induced by the DTM effect. Analysis of successive F2 plants indicated that the mutations induced by the DTM effect were largely heritable. Moreover, DTM-generated hybrids had significantly smaller leaf angles that were reduced more than 50% when compared with that of the wild type. Planting experiments showed that DTM-generated maize plants can be grown with significantly higher density and hence greater yield potential. Our work demonstrate that stably expressed RGEN could be implemented as an in vivoDTM to rapidly generate and spread desired mutations in maize through hybridization and subsequent backcrossing, and hence bypassing the linkage drag effect in convention introgression methodology. This proof-of-concept experiment can be a potentially much more efficient breeding strategy in crops employing the RNA-guided Cas9 genome editing.