Class 2 CRISPR systems are exceptionally diverse, nevertheless, all share a single effector protein that contains a conserved RuvC-like nuclease domain. Interestingly, the size of these CRISPR-associated (Cas) nucleases ranges from >1000 amino acids (aa) for Cas9/Cas12a to as small as 400-600 aa for Cas12f. For in vivo genome editing applications, compact RNA-guided nucleases are desirable and would streamline cellular delivery approaches. Although miniature Cas12f effectors have been shown to cleave double-stranded DNA, targeted DNA modification in eukaryotic cells has yet to be demonstrated. Here, we biochemically characterize two miniature type V-F Cas nucleases, SpCas12f1 (497 aa) and AsCas12f1 (422 aa), and show that SpCas12f1 functions in both plant and human cells to produce targeted modifications with outcomes in plants being enhanced with short heat pulses. Our findings pave the way for the development of miniature Cas12f1-based genome editing tools.

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.

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 (DSBs) 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 were observed at the donor loci and the genomic target site, respectively. Unlike previous in planta or intra-genomic homologous recombination reports, that required multiple generations and extensive screening, our method provides non-chimeric, heritable GT in the T0 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 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.