Agrobacterium-Mediated Transformation and Targeted Mutagenesis Using SpCas12f in Rice

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR-Cas) is an adaptive prokaryote immune system against foreign DNA/RNA that is now applied widely to genome editing. A miniature Cas, CRISPR-Cas12f, is one-half to one-third the size of the CRISPR-Cas9 that is commonly used in genome editing experiments in many organisms, including higher plants. The compactness of CRISPR-Cas12f is expected to be advantageous in terms of vector construction and transformation frequency. Moreover, CRISPR-Cas12f can be useful for virus vector-mediated genome editing because the size of the transgene is the major restriction in the use of virus vectors. Here, we describe our protocol for targeted mutagenesis using Cas12f derived from Syntrophomonas palmitatica (SpCas12f) via Agrobacterium-mediated transformation in rice. We also summarize some approaches to improve the frequency of targeted mutagenesis using SpCas12f.