Enhancing Cas12a and Cas9 mediated plant genome editing efficiency and deletion size with the sbcB exonuclease

CRISPR-Cas9 and Cas12a stand as two prominent genome editing systems widely used in plants. In recent years, considerable efforts have been dedicated to enhancing their genome editing efficiency. However, equally significant is the endeavor to improve these CRISPR systems by altering their genome editing profiles. Constrained by their modes of action, Cas9 and Cas12a typically generate small deletions in plant cells. To expand the capabilities of these nucleases to generate significantly large deletions, we tested a series of exonucleases via direct fusion to the Cas protein. Based on testing in rice protoplasts and stable lines, we observed that the fusion of Cas9 and LbCas12a with sbcB exonuclease from Escherichia coli induced larger deletions. Equipped with this unique capability, sbcB-zSpCas9 and sbcB-LbCas12a will be powerful tools to knock out non-coding genes, investigate cis-regulatory elements, and engineer quantitative traits in plants.