Engineering Introns to Express RNA Guides for Cas9- and Cpf1-Mediated Multiplex Genome Editing  Dan Ding, Kaiyuan Chen, Yuedan Chen, Hong Li, Kabin Xie  Molecular Plant  Volume 11, Issue 4, Pages 542-552 (April 2018) DOI: 10.1016/j.molp.2018.02.005 Copyright © 2018 The Author Terms and Conditions

Figure 1 Engineering the Intron to Express Polycistronic tRNA-gRNA (PTG) with Cas9 for Multiplex Genome Editing. (A) Schematic illustration of intron splicing from primary mRNA. (B) Schematics of PTG processing to release individual gRNAs. The endogenous RNase P and Z specifically recognize and precisely cut the tRNA unit in the PTG transcript to release gRNAs. (C) Schematic depiction of the strategy to engineer the intron to express gRNAs for multiplex genome editing. After integrating the PTG between the donor and branch sites of an intron, the gRNAs are cut out from the spliced intron by endogenous RNase P and Z, while the mature mRNA is translated as normal. The intronic PTG can be fused with Cas9 coding sequence (inPTG-Cas9) as normal intron and exon, which permits expression of all CRISPR components in one gene. Molecular Plant 2018 11, 542-552DOI: (10.1016/j.molp.2018.02.005) Copyright © 2018 The Author Terms and Conditions

Figure 2 The inPTG-Cas9 Is Robust and Efficient for Multiplex Genome Editing in Rice Protoplasts. (A) Schematic structures of UBI10 gene, conventional U3p::PTG-UBI10p::Cas9 (pRGEB32), and UBI10p::inPTG-Cas9 vectors (pRGEB33 and pRGEB34). Boxes/lines indicate exons/introns, and triangles represent the dual-BsaI cloning sites, whose sequences are shown at the bottom of the plot. The BsaI sites are highlighted in red. Small arrows indicate the position of RT–PCR primers. (B) Structure and targets of synthetic inPTG fragments. Each gene was targeted by a pair of gRNAs tandemly arrayed with tRNA in inPTGs. (C) RT–PCR amplified splicing products from UBI10p::Cas9 (pRGEB32) and UBI10p::inPTG-Cas9 (pRGEB33 and pRGEB34). (D) The representative sequence chromatograph of RT–PCR products, confirming that inPTG-Cas9 was spliced at original donor and acceptor sites (red letters). (E) Protein level of Cas9 expressed with UBI10p::Cas9 (pRGEB32) and UBI10p::inPTG-Cas9 (pRGEB33 and pRGEB34). The loading of total protein was shown by Coomassie brilliant blue staining. (F and G) PCR detection of the chromosomal fragment deletions (Del) at targeted loci in rice protoplasts expressing respective inPTG-Cas9. The conventional U3p::PTG-UBI10p::Cas9 constructs and empty vector were used as controls. U3p, snoRNA U3 promoter; UBI10p, UBI10 promoter; CK, empty vector control; M, DNA marker; WT, wild-type; RT, reverse transcriptase. Molecular Plant 2018 11, 542-552DOI: (10.1016/j.molp.2018.02.005) Copyright © 2018 The Author Terms and Conditions

Figure 3 Targeted Mutation Frequency in Stable Rice Plants Expressing inPTG-Cas9. (A) The albino seedlings from inPTG10-Cas9 transgenic plants (T0 generation). (B) Indel frequency of inPTG3/6/7/10-Cas9 transgenic plants. The editing frequency was examined using PCR/RE assays with subsequent confirmation by DNA sequencing (see also Supplemental Figures 6–9). Vec, empty vector control. Molecular Plant 2018 11, 542-552DOI: (10.1016/j.molp.2018.02.005) Copyright © 2018 The Author Terms and Conditions

Figure 4 The inPTG Can Be Integrated at Different Positions and Expressed with Various Pol II Promoters. (A) Structure of the Cas9-inPTG construct (pRGEB33T). The PTG was inserted at the 3′ UTR intron at the dual-BsaI site (indicated by a triangle). (B) Target fragment deletion efficiency (Del %) of Cas9-inPTG7 at MPK1 and MPK5 loci using different PTG expression cassettes. (C) Schematic structure of PR1p::inPTG-Cas9 and PR5p::inPTG-Cas9 vectors. Boxes/lines indicate exons/introns, and triangles represent the dual-BsaI cloning site. (D) Target fragment deletion at MPK2 gene using PR1p- and PR5p-expressed inPTG4-Cas9 in rice protoplasts. Molecular Plant 2018 11, 542-552DOI: (10.1016/j.molp.2018.02.005) Copyright © 2018 The Author Terms and Conditions

Figure 5 Engineering Introns to Express crRNAs for Cpf1-Mediated Genome Editing. (A) Schematic illustration of Cpf1-mediated genome editing. Left: DNA targeting with Cpf1 and crRNA. The crRNA consists of 5′-end direct repeat (DR, stem-loop region) and 3′-end guide sequence (spacer). Right: Cpf1 possesses RNase activity to cleave the crRNA array. The Cpf1 cleaved site is indicated by scissors. (B) Structures of plasmid vectors to express Cpf1 and crRNAs. The U3p (p32Lb and p32Fn) and intron (p33Lb and p33Fn) were used to express crRNAs. The crRNAs are organized as polycistronic tRNA-crRNA (PTC) or crRNA array (CA) and inserted at the dual-BsaI cloning site indicated by a triangle. (C) A pair of crRNAs were designed to target the rice PDS gene. (D) Schematic structure of PTCPDS and CAPDS. (E) Comparison of target fragment deletion frequency (Del %) using different crRNA expression cassettes with FnCpf1 and LbCpf1 (shown as Fn and Lb). (F) Western blotting shows the Cpf1 protein level expressed with different constructs. The loading of total protein is shown at the bottom by Coomassie brilliant blue staining. Fn, FnCpf1; Lb, LbCpf1; CK, empty vector control. Molecular Plant 2018 11, 542-552DOI: (10.1016/j.molp.2018.02.005) Copyright © 2018 The Author Terms and Conditions

Figure 6 Simultaneous Editing of MPK2 and MPK5 Using Cpf1 and crRNA Arrays. (A) Schematic illustrations of targeting sites in MPK2 and MPK5. A pair of crRNAs was designed for each gene, and the PAM sequence is shown in red. (B) Structure of crRNA array (CAMPK) to simultaneously target MPK2 and MPK5. (C) Comparison of targeted fragment deletion frequencies (Del) using different CAMPK expression cassette with FnCpf1 and LbCpf1. DR, direct repeat of crRNA; Fn, FnCpf1; Lb, LbCpf1; M, DNA markers; CK, empty vector control. Molecular Plant 2018 11, 542-552DOI: (10.1016/j.molp.2018.02.005) Copyright © 2018 The Author Terms and Conditions