NEB [1] Plasmids pdest-hismbp-ascpf1-ec E. coli codon optimized AsCpf1 coding sequence, Am R [2] pmtl82151 pbp1 ori, Cm R, ColE1 ori, TraJ [3]

Transcription

1 Table S1 Bacterial strains and plasmids used in this study. Strains or Plasmids Relevant characteristics Reference or source Strains E. coli NEB Express Competent E. coli (High efficiency) fhua2 [Ion] ompt gal sula11 R(mcr-73::miniTn10--Tet S )2[dcm]R(zgb-210::Tn10--Tet S )enda delta(mcrc-mrr)114::is10 E. coli CA434 hsd20(r B -, m B -), reca13, rpsl20, leu, proa2, with IncPb conjugative plasmid R702 Clostridium difficile C. difficile 630 WT stain From Dr. Shonna McBride (Emory University) fur Derived from C. difficile 630, fur tetm Derived from C. difficile 630, tetm ermb1/2 Derived from C. difficile 630, ermb1/2 cwp66 tcda Derived from C. difficile 630, cwp66 tcda ermb1/2 tetm Derived from ermb1/2, ermb1/2 tetm phicd630-2 Derived from C. difficile 630, phicd630-2 NEB [1] Plasmids pdest-hismbp-ascpf1-ec E. coli codon optimized AsCpf1 coding sequence, Am R [2] pmtl82151 pbp1 ori, Cm R, ColE1 ori, TraJ [3] pwh34 Derived from pmtl82151, E. coli-c. difficile shuttle vector, ilacp::ascpf1, BtgZI-BtgZI double sites, Chassis plasmid for 1

2 pwh37 pwh41s pwh41 pwh55 pwh74 pwh75 pwh79 pjz173 pjz174 pyw34-btgzi pjz23-cas9 pjz180 gene-targeting plasmid construction, Cm R /Tm R Derived from pwh34, ilacp::ascpf1, srnap::crrna (23-nt repeat), Cm R /Tm R, PCR template for generating srnap::crrna for retargeting Derived from pwh34, targeting Ferric-uptake regulator (fur) gene, without homology arms, Cm R /Tm R Derived from pwh34, targeting Ferric-uptake regulator (fur) gene, with homology arms, Cm R /Tm R Derived from pwh34, targeting cell wall protein cwp66 (cwp66) gene, Cm R /Tm R Derived from pwh34, targeting tetracycline resistance protein Tn5397 encoding (tetm) gene, Cm R /Tm R Derived from pwh34, targeting ermb1, ermb2 genes (ermb1/2), Cm R /Tm R Derived from pwh34 and pwh55, targeting cwp66 and tcda genes, Cm R /Tm R Derived from pwh34, targeting phicd630-2 with one spacer, Cm R /Tm R Derived from pwh34, targeting phicd630-2 with two sets of spacers, Cm R /Tm R General CRISPR-Cas9 vector, same as pyw34-pta, but without the 20-nt protospacer sequence; with two BtgZI sites for easy re-targeting purposes. pyw34-btgzi derivative; pbp1 ori, ColE1 ori, Amp R, Cm R, TraJ, ilacp-cas9, grna Derived from pjz23-cas9, E. coli-c. difficile shuttle vector, targeting phicd630-2, Cm R /Tm R 2 [4]

3 3

4 Table S2 Primers used in this study. Primers Sequences (5-3 ) pwh34 YW2817 YW2818 YW2819 YW2820 YW2823 YW2824 pwh37 YW2821 YW2822 pwh41s YW3105 YW3106 pwh41 YW3105 YW3713 YW3714 YW3715 YW3716 YW3717 YW3718 YW3719 pwh55 YW3304 CAGCTATGACCGCGGCCGCTGTATCTTATATACTTGGTTTATTTACTTGA TGGTGAAGCCTTCAAACTGTGTCATTTCAGCCCTCCTGTGAAATTGTTAT ATAACAATTTCACAGGAGGGCTGAAATGACACAGTTTGAAGGCTTCACCA GAGCTCGAATTCGTAATCATGGTTTAGTTTCTCAGTTCTTGAATGTAGGCC TACGAATTCGAGCTCGGTACCCGGGTATAAATTAAGATTTAAAAAGGTTA CGTGACGTCGACTCTAGATGCTTGCAGTCATCGCTGACTCATGCGATGAGCTAGGTCAATTTCTATTTTCTTCTTTTAAC TACGAATTCGAGCTCGGTACCCGGGTATAAATTAAGATTTAAAAAGGTTA CGTGACGTCGACTCTAGATATTAAGTCCTTCTTATCAGATGATCTACAAGAGTAGAAATTAATGGTGGAATGATAAGGGT AAAGTTAAAAGAAGAAAATAGAAATATAATCTTTAATTTGAAAAGATTTA CTCCATGGACGCGTGACGTCGACTCATCTACAAGAGTAGAAATTAAAGCCATGTTCCGTTAGCTGAGC AAAGTTAAAAGAAGAAAATAGAAATATAATCTTTAATTTGAAAAGATTTA ATTAAAGCCATGTTCCGTTAGCTGAGCATCTACAAGAGTAGAAATTAATGGTGGAATGATAAGGGTT AGATGCTCAGCTAACGGAACATGGCTTTAATTTCTACTCTTGTAGATGCAGGAGAGTCAGAAGCAAGAAATA ATTAATTATTTTTAGAATTTGAAAAAGTTAACATTCCTTTTTAATAATGA TCATTATTAAAAAGGAATGTTAACTTTTTCAAATTCTAAAAATAATTAAT CTCCATGGACGCGTGACGTCGACTCCAGGGCATTCCCAATCTTCACTTAA ATAGCGACAAGAGAGAGTAC CTTCACCACTACACATAGCTG ATTATTAGCTGCTAATACACCCACTGCATCTACAAGAGTAGAAATTAATGGTGGAATGATAAGGGTT 4

5 5 YW3305 YW3306 YW3307 YW3308 YW2369 YW2370 AGATGCAGTGGGTGTATTAGCAGCTAATAATTTCTACTCTTGTAGATTAGCTGTGAAGCAATGGGTGTAAAA TTTTGTTTTTAAAAGATTTTAATATTAATTCTTACCTCCTAATTCTTTCG CGAAAGAATTAGGAGGTAAGAATTAATATTAAAATCTTTTAAAAACAAAA CTCCATGGACGCGTGACGTCGACTCGCTGAGACAGGTATTGTTGACACTA TGGAGGGTCATGTTCTTATC GCTGAGACAGGTATTGTTGACA pwh74 YW3456 YW3457 YW3458 YW3459 YW3460 YW3461 YW3462 ATTATCCGATATTCTAACCGAATCCCGATCTACAAGAGTAGAAATTAATGGTGGAATGATAAGGGTT AGATCGGGATTCGGTTAGAATATCGGATAATTTCTACTCTTGTAGATTATCCTACAGCAACAGCGAGTGAAC CATTTATATAACAATATAAAATGTGGTGATTTTCCTCCATTCAAAAGCCA TGGCTTTTGAATGGAGGAAAATCACCACATTTTATATTGTTATATAAATG CTCCATGGACGCGTGACGTCGACTCGCAACACCCAAGCATACAAGTCCTC GTGATGGTACAGTTGATTCC GAGATTGACCCACGTTACCTAA pwh75 YW3463 YW3464 YW3465 YW3466 YW3467 YW3468 YW3469 ATTATAGATGTCAGACGCACGGCTTTCATCTACAAGAGTAGAAATTAATGGTGGAATGATAAGGGTT AGATGAAAGCCGTGCGTCTGACATCTATAATTTCTACTCTTGTAGATGCAGGGTTACTTATTGGAACAGTGT AAATTTAAAAAAGCGACTCATAGAAGTAATCACTCCTGAAGTGATTACAT ATGTAATCACTTCAGGAGTGATTACTTCTATGAGTCGCTTTTTTAAATTT CTCCATGGACGCGTGACGTCGACTCCCCACAATCACTCACTTCTTTCTAT ACGAACCGTCTTATCTCCCA CTCTTTCCTTAGAGTACGTC pwh79 YW3529 YW3530 YW3531 YW3532 AAGAGTAGAAATTAAGTTGAACCTTAACTGAGGTACTATCTACAAGAGTAGAAATTATTAGCTGCTAATACACCCACTGC TTTCTACTCTTGTAGATAGTACCTCAGTTAAGGTTCAACTTAATTTCTACTCTTGTAGATTAGCTGTGAAGCAATGGGTG ATAGATTTTACCAACTGACCATGCACCATCGCTGAGACAGGTATTGTTGACACTATTAAA TTTAATAGTGTCAACAATACCTGTCTCAGCGATGGTGCATGGTCAGTTGGTAAAATCTAT

6 6 YW3533 YW3534 YW3535 YW3321 YW3322 ACAGGAATAATTTTTTATCAAACATATATTAAAAACCTCCTAGTATTATTATTTTTGATA TATCAAAAATAATAATACTAGGAGGTTTTTAATATATGTTTGATAAAAAATTATTCCTGT GAGATCTCCATGGACGCGTGACGTCGACTCGAAGATGGTGATGAGGTGCAAATAAGATTA GATGCCAAGAGACACACAAG CCATGAGGAGGTCATTTCTAACCA pjz173 YW3333 YW3334 YW3335 YW3336 YW3337 YW3342 YW3343 YW3799 YW3856 TTAGGAGTGACTTTGAGCTTGATGTAATCTACAAGAGTAGAAATTAATGGTGGAATGATAAGG GATTACATCAAGCTCAAAGTCACTCCTAATTTCTACTCTTGTAGATGACCTATTGCATGCATCATTG GCACATGAAGTAGCAGTTACTTCCCAGTTGGATTGACTAATGAAGCTC GAGCTTCATTAGTCAATCCAACTGGGAAGTAACTGCTACTTCATGTGC CTCCATGGACGCGTGACGTCGACTCTGAAGGTTAAGGATATGGTGCT ATTGGAACATGCACTCCAAGT GCACAGCTTCTTGCACAAGAG AGTGGAACTATAATTCTCCAGGAT GCTTGTGTCTAACCTTTGCATCT pjz174 YW3338 YW3339 YW3335 YW3336 YW3337 TTAGTGGTACAATTCCCATAGGTTACATCTACAAGAGTAGAAATTAGGAGTGACTTTGAGCTTGATGT GATGTAACCTATGGGAATTGTACCACTAATTTCTACTCTTGTAGATGACCTATTGCATGCATCATTG GCACATGAAGTAGCAGTTACTTCCCAGTTGGATTGACTAATGAAGCTC GAGCTTCATTAGTCAATCCAACTGGGAAGTAACTGCTACTTCATGTGC CTCCATGGACGCGTGACGTCGACTCTGAAGGTTAAGGATATGGTGCT pjz180 YW1029 ACAATTGAATTTAAAAGAAACCGATAGGCCGGCCAGTGGGCAAGTTG YW1030 CTTTAGTAACGTGTAACTTTCCAAATGGAGTTTAAACTTAGGGTAAC YW1073 CGAACACGAACCGTCTTATCTCCCATTGTTCTGAATCCTTAGCTAATGG YW1074 TAATGACCCCGAAGCAGGGGGCCCAATGAATTTGTAAATAAACCACAAAC YW1060 GTAATACTAAAACTGAATTGATTCCTGCTTCGGGGTCATTATAG

7 YW1061 YW3374 YW3375 YW3335 YW3336 YW3376 ATCAAGTAAATAAACCAAGTATATAAGGGCCCGATCGGTCTTGCCTTGCTCGTCG TTGCTATTTCTAGCTCTAAAACGCAGCAGTGCTAGAATCGTAATGGTGGAATGATAAGGG CTTTGTGATATGACTAATAATTAGCGGCCGCGACCTATTGCATGCATCATTG GCACATGAAGTAGCAGTTACTTCCCAGTTGGATTGACTAATGAAGCTC GAGCTTCATTAGTCAATCCAACTGGGAAGTAACTGCTACTTCATGTGC ATCCACTAGTAACCATCACACTGGCGGCCGCTGAAGGTTAAGGATATGGTGCT Supplementary References [1] D.R. Williams, D.I. Young, M. Young, Conjugative plasmid transfer from Escherichia coli to Clostridium acetobutylicum, J. Gen. Microbiol. 136 (1990) doi: / [2] J.K. Hur, K. Kim, K.W. Been, G. Baek, S. Ye, J.W. Hur, S. Ryu, Y.S. Lee, J. Kim, Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins Generation of knockout mice by Cpf1-mediated gene targeting, Nat. Publ. Gr. 34 (2016) doi: /nbt [3] J.T. Heap, O.J. Pennington, S.T. Cartman, N.P. Minton, A modular system for Clostridium shuttle plasmids, J. Microbiol. Methods. 78 (2009) doi: /j.mimet [4] Y. Wang, Z.T. Zhang, S.O. Seo, P. Lynn, T. Lu, Y.S. Jin, H.P. Blaschek, Bacterial Genome Editing with CRISPR-Cas9: Deletion, Integration, Single Nucleotide Modification, and Desirable clean Mutant Selection in Clostridium beijerinckii as an Example, ACS Synth. Biol. 5 (2016) doi: /acssynbio.6b