Figure 1. AsCpf1 and LbCpf1-mediated gene editing in human cells

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Figure 1. AsCpf1 and LbCpf1-mediated gene editing in human cells Figure 1. AsCpf1 and LbCpf1-mediated gene editing in human cells. (A) Illustration of the GFP-reporter system for measuring Cpf1-mediated DNA cleavage in human cells. Cpf1-family proteins were generated with the corresponding plasmids expressed in 293-SC1 cells. The corresponding crRNA was expressed droved with U6 promoter. Knock-out efficiency was analyzed using flow cytometry. (B) Schematic diagram of the AsCpf1 crRNA-DNA-targeting complex. The target sequence is shown in blue and the PAM sequence is shown in pink. (C) AsCpf1 and LbCpf1 recognized 5′-TTTN-3′ PAMs. AsCpf1 and LbCpf1 failed to cleave target sites with a 5′-GCTN-3′ PAM or 5′-CATN-3′ PAM, respectively. Error bars, S.E.M.; n = 3; NC, negative control. **P < 0.01. From: A ‘new lease of life’: FnCpf1 possesses DNA cleavage activity for genome editing in human cells Nucleic Acids Res. Published online September 07, 2017. doi:10.1093/nar/gkx783 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 2. FnCpf1 possesses DNA cleavage activity in human cells Figure 2. FnCpf1 possesses DNA cleavage activity in human cells. (A) Sequence of GFP target site 2. (B) Cells were co-transfected with plasmids encoding FnCpf1 and plasmids for crRNA expression target site 2. (C) DNA sequence chromatograms of the fragments harboring the target site obtained from the cells treated with FnCpf1 are in a mass, compared with controls. (D) DNA sequence analysis of single individual GFP-negative colonies. Dashes represent the DNA deletions. The number at the right side of each sequence is the length of indel (−, deletion). (E) Comparison of the activity of FnCpf1 with that of AsCpf1 and LbCpf1. Cells was co-transfected with plasmids encoding Cpf1 orthologs (FnCpf1, AsCpf1 and LbCpf1) and plasmids encoding crRNAs target site 1 in various combinations. Error bars, S.E.M.; n = 3; **P < 0.01. From: A ‘new lease of life’: FnCpf1 possesses DNA cleavage activity for genome editing in human cells Nucleic Acids Res. Published online September 07, 2017. doi:10.1093/nar/gkx783 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 4. On-target gene editing activities of crRNA with different length of spacer sequence and direct repeat in human cells. (A, B) Lengths and sequences of crRNA spacer regions are shown. Indel frequencies were measured by flow cytometry. The two target sites used are named as previously described. The spacer sequence for FnCpf1 at 21nt could lead to maximum gene editing efficiency. (C) Alignment of direct repeat sequences from the 16 member Cpf1 family. The stem structure is highlighted in gray. Non-conserved sequences are in red. The direct repeats sequence of PcCpf1 (14) is identical with PmCpf1(P.macacae.Cpf1). (D) Cells were co-transfected with plasmids encoding FnCpf1 and plasmids for crRNA expression, of which direct repeats are from 16 Cpf1 members. Error bars, S.E.N.; n = 3; *P < 0.05, **P < 0.01. From: A ‘new lease of life’: FnCpf1 possesses DNA cleavage activity for genome editing in human cells Nucleic Acids Res. Published online September 07, 2017. doi:10.1093/nar/gkx783 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 3. FnCpf1 induced indel mutations on endogenous genes Figure 3. FnCpf1 induced indel mutations on endogenous genes. (A, C, E) Cleaved amplified polymorphic sequences (CAPS) analysis of DNMT1–1, RS1 and NRL loci. Arrow indicated the position of digestion-resistant PCR products. Digestion-resistant bands are the fragment with the indel mutation. Molecular markers in A, C and E are DL2000 (TaKaRa). (B, D, F) Restriction Enzyme cutting site (Pst1, Xho1) are shown in the target DNA sequences of each loci. Sequencing reads show representative mutations of Fncpf1 mediated gene editing with its own crRNA in DNMT1–1, RS1 and NRL loci. Dashes represent the DNA deletions. The number at the right side of each sequence is the length of indel (−, deletion). From: A ‘new lease of life’: FnCpf1 possesses DNA cleavage activity for genome editing in human cells Nucleic Acids Res. Published online September 07, 2017. doi:10.1093/nar/gkx783 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 5. PAMs for FnCpf1-mediated gene editing in human cells Figure 5. PAMs for FnCpf1-mediated gene editing in human cells. (A) Schematic diagram of targeted sites with different PAMs in GFP. Target sites and PAM sequences are in blue and purple, respectively. The target sequences were show in Supplementary Table S2. (B) Among these 16 PAMs (NNn), CTC PAM has the relative highest level of FnCpf1 mediated DNA cleavage expect TTN(TTA). (C, D) FnCpf1 recognizes a PAM, defined as 5′-YTV-3′. NC, negative control. Error bars, S.E.M.; n = 3; *P < 0.05, **P < 0.01. From: A ‘new lease of life’: FnCpf1 possesses DNA cleavage activity for genome editing in human cells Nucleic Acids Res. Published online September 07, 2017. doi:10.1093/nar/gkx783 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 6. Specificities of FnCpf1 with different crRNAs Figure 6. Specificities of FnCpf1 with different crRNAs. (A, B) The activity of single-nucleotide mismatched crRNAs. (C) Effect of crRNA-target DNA match or mismatch on FnCpf1 cleavage activity. NC, negative control. Error bars, S.E.M.; n = 3; From: A ‘new lease of life’: FnCpf1 possesses DNA cleavage activity for genome editing in human cells Nucleic Acids Res. Published online September 07, 2017. doi:10.1093/nar/gkx783 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com