Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing

CRISPR‐Cas9 and Cas12a are two powerful genome editing systems. Expression of CRISPR in plants is typically achieved with a mixed dual promoter system, in which Cas protein is expressed by a Pol II promoter and a guide RNA is expressed by a species‐specific Pol III promoter such as U6 or U3. To achieve coordinated expression and compact vector packaging, it is desirable to express both CRISPR components under a single Pol II promoter. Previously, we demonstrated a first‐generation single transcript unit (STU)‐Cas9 system, STU‐Cas9‐RZ, which is based on hammerhead ribozyme for processing single guide RNAs (sgRNAs). In this study, we developed two new STU‐Cas9 systems and one STU‐Cas12a system for applications in plants, collectively called the STU CRISPR 2.0 systems. We demonstrated these systems for genome editing in rice with both transient expression and stable transgenesis. The two STU‐Cas9 2.0 systems process the sgRNAs with Csy4 ribonuclease and endogenous tRNA processing system respectively. Both STU‐Cas9‐Csy4 and STU‐Cas9‐tRNA systems showed more robust genome editing efficiencies than our first‐generation STU‐Cas9‐RZ system and the conventional mixed dual promoter system. We further applied the STU‐Cas9‐tRNA system to compare two C to T base editing systems based on rAPOBEC1 and PmCDA1 cytidine deaminases. The results suggest STU‐based PmCDA1 base editor system is highly efficient in rice. The STU‐Cas12a system, based on Cas12a’ self‐processing of a CRISPR RNA (crRNA) array, was also developed and demonstrated for expression of a single crRNA and four crRNAs. Altogether, our STU CRISPR 2.0 systems further expanded the CRISPR toolbox for plant genome editing and other applications.