Expanding the Scope of CRISPR/Cpf1-Mediated Genome Editing in Rice Shaoya Li, Xin Zhang, Wensheng Wang, Xiuping Guo, Zhichao Wu, Wenming Du, Yunde Zhao, Lanqin Xia  Molecular Plant  Volume 11, Issue 7, Pages 995-998 (July 2018) DOI: 10.1016/j.molp.2018.03.009 Copyright © 2018 The Author Terms and Conditions

Figure 1 Editing Rice Genes by LbCpf1 Variants. (A) Linearized CRISPR/Cpf1 constructs. The constructs produce LbCpf1 RR variant and crRNAs targeting OsPDS, OsSBEIIb, or both OsPDS and OsSBEIIb. LbCpf1(RR) was inserted downstream of the ZmUbi promoter. HH and HDV are ribozyme units. The two crRNA ribozyme units (RCR1 and RCR2) are under the control of OsU3 promotor. Upon transcription, the two crRNAs can be released by self-cleavage. (B) PCR/T7E1 assay of representative editing events in OsPDS sites. PCR products amplified by primers T7E1-PDSF/R were digested with T7E1. Lines 17 and 67 only had mutations around the target PDS1. Line 17 had a heterozygous 79 bp deletion. Line 67 was chimeric with three alleles: a 10 bp deletion, a 38 bp deletion, and wild-type. Line 21 only had mutations around the target PDS2 with a heterozygous 10 bp deletion. In line 34, both target sites were mutated and contained a heterozygous 287 bp deletion between the two targets. (C) Summary of targeted mutagenesis generated by LbCpf1 RR variant in rice T0 plants. Bi, bi-allele; He, heterozygote; Chi, Chimera. (D) PCR/T7E1 assay of representative editing events in OsSBEⅡb targets. PCR products amplified by primers T7E1-SBEⅡbF/R were digested with T7E1. Lines 22, 41-7, and 54 only had mutations around the target SBEⅡb1. Line 22 had a heterozygous 87 bp deletion. The line 41-7 was bi-allelic with one 9 bp deletion and one 86 bp deletion. The line 54 was chimeric with three alleles: a 7 bp deletion, a 37 bp deletion, and wild-type. In line 41-4, both target sites were mutated. One allele had a 37 bp deletion around target SBEⅡb1 and a 19 bp deletion around target SBEⅡb2, while another one was wild-type. (E) Analysis of editing events in both OsPDS and OsSBEⅡb loci. PCR products amplified by primers T7E1-PDSF/R and T7E1-SBEⅡbF/R were digested with T7EI. Line 53 only had mutations around target PDS1. This line had one allele with a 13 bp deletion, while another one was wild-type. Lines 12 and 56 only had mutations around the target SBEⅡb1. Line 12 was a chimeric line with three alleles, one allele had a 9 bp deletion, another allele had a 7 bp deletion, while the third allele was wild-type. Line 56 was a bi-allelic line: one 16 bp deletion and a 7 bp deletion. In line 62, two target sites were mutated simultaneously. One allele had a 15 bp deletion and a 10 bp insertion around target PDS1 and a 12 bp deletion in target SBEⅡb1, while another one was wild-type. (F) Potentially targetable sites in rice for LbCpf1 and its RR variant. Computational analysis of the rice genome sequence (Os-Nipponbare-Reference-IRGSP-1.0) revealed that 96.0% of genes have the TTTV PAMs and 99.6% harbor the TYCV PAMs. The RR variant increased the targetable sites by CRISPR/Cpf1 to 99.7% of rice genes. PAM/guide sequences are marked in gray and PAM (TYCV) are marked in red and underlined. T7E1, T7 endonuclease I, which cannot cut PCR products from WT; M, DNA marker DL2000; WT, wild-type. Molecular Plant 2018 11, 995-998DOI: (10.1016/j.molp.2018.03.009) Copyright © 2018 The Author Terms and Conditions