Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing.

The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy.

Establishment Limitation Constrains the Abundance of Lactic Acid Bacteria in the Napa Cabbage Phyllosphere.

Patterns of phyllosphere diversity have become increasingly clear with high-throughput sequencing surveys, but the processes that control phyllosphere diversity are still emerging. Through a combination of lab and field experiments using Napa cabbage and lactic acid bacteria (LAB), we examined how dispersal and establishment processes shape the ecological distributions of phyllosphere bacteria. We first determined the abundance and diversity of LAB on Napa cabbage grown at three sites using both culture-based approaches and 16S rRNA gene amplicon sequencing. Across all sites, LAB made up less than 0.9% of the total bacterial community abundance. To assess whether LAB were low in abundance in the Napa cabbage phyllosphere due to a limited abundance in local species pools (source limitation), we quantified LAB in leaf and soil samples across 51 vegetable farms and gardens throughout the northeastern United States. Across all sites, LAB comprised less than 3.2% of the soil bacterial communities and less than 1.6% of phyllosphere bacterial communities. To assess whether LAB are unable to grow in the phyllosphere even if they dispersed at high rates (establishment limitation), we used a gnotobiotic Napa cabbage system in the lab with experimental communities mimicking various dispersal rates of LAB. Even at high dispersal rates, LAB became rare or completely undetectable in experimental communities, suggesting that they are also establishment limited. Collectively, our data demonstrate that the low abundance of LAB in phyllosphere communities may be explained by establishment limitation.IMPORTANCE The quality and safety of vegetable fermentations are dependent on the activities of LAB naturally present in the phyllosphere. Despite their critical role in determining the success of fermentation, the processes that determine the abundance and diversity of LAB in vegetables used for fermentation are poorly characterized. Our work demonstrates that the limited ability of LAB to grow in the cabbage phyllosphere environment may constrain their abundance on cabbage leaves. These results suggest that commercial fermentation of Napa cabbage proceeds despite low and variable abundances of LAB across different growing regions. Propagule limitation may also explain ecological distributions of other rare members of phyllosphere microbes.