Chapter 19 Editing a Stomatal Developmental Gene in Rice with CRISPR/Cpf1 Xiaojia Yin, Abhishek Anand, Paul Quick, and Anindya Bandyopadhyay Abstract CRISPR has arguably been the fastest growing genome editing tool so far. CRISPR/Cas9 (Cas9) has been proved to be efficient and precise in genome editing. However Cas9 has certain limitations. CRISPR/Cpf1 (Cpf1) has been discovered as an alternate approach that can overcome some of those limitations. Cpf1 allows targeting in AT-rich region, creating a staggered cleavage, and cutting at the distal end to the PAM (Protospacer Adjacent Motif) regions. We have successfully tested the efficiency of Cpf1 system in rice using OsEPFL9 which is a developmental gene known to regulate the stomatal density in leaf. Regulation of stomatal density and patterning is an important factor in regulating plant physiology, especially in improv- ing the plant water use efficiency. We targeted the Exon1 of OsEPFL9 and the knockout lines were studied for several generations for establishment of stabilized editing, as well as transmission and segregation of edits through generations. The usage of Cpf1 as a genome editing tool to manipulate stomatal patterning may further help us gain more insight of the physiology of rice in stress conditions. Key words CRISPR/Cpf1, OsEPFL9, Genome editing, Rice 1 Introduction Appearance of stomata was one of the most significant traits in the course of plant evolution that helped plants ascend from aqueous environment to adapt and conquer the land [1]. Stomata are pres- ent in almost all land plants [2]. The appearance of stomata dates back to the similar period as the evolution of land plants began. Although their distribution and patterning vary significantly, pri- marily depending on the habitat of the plants as an adaptive mea- sure, there has been only little change in the stomatal structure since its evolution [2]. However this does not hold true for the developmental stages of the stomata, as the developmental stages have become much more complicated involving several pathways and regulatory factors [3–5]. Yiping Qi (ed.), Plant Genome Editing with CRISPR Systems: Methods and Protocols, Methods in Molecular Biology, vol. 1917, https://doi.org/10.1007/978-1-4939-8991-1_19, © Springer Science+Business Media, LLC, part of Springer Nature 2019 Xiaojia Yin and Abhishek Anand have contributed equally to this work. 257