Fig. 1.
Design and optimization of the Cpf1-gRNAtRNA expression structure. a Secondary cloverleaf structure of the eukaryotic pre-tRNA. Acceptor stem is colored in purple, variable loop in orange, D arm in brown, T arm in green, anticodon-loop in blue, anticodon in black, 5′ leader and 3′ tailor in blank circles. This pre-tRNA can be cleaved by RNase P and RNase Z at specific sites. b Structure of Cpf1 gRNAtRNA system with U6/T7 promoter-gRNA-tRNA. c One gRNA for Cas9 and one gRNA for Cpf1 were designed and assembled to target the hROSA26-EGFP (green highlight). Red letters indicate the sequence of target site, and green letters indicate the PAM site. d EGFP disruption rates of 43 different gRNAtRNA with Cpf1 shows that pre-tRNAGln−TTG−1−1 featured the highest gene editing efficiency