Supplemental Figure S1. Diagram of PCR fragments utilized to complement or mutate CRISPR2. CRISPR2 schematic representation depicts repeat sequences as black lines, deleted sequence as dotted lines, Wt spacers of PA14 CRISPR2 as grey boxes, P. aeruginosa strain 2192 CRISPR1 spacer 8 as black boxes, and point mutations within spacer 1 as white boxes. (A)Diagram of wt CRISPR2 and flanking DNA. (B) Amplification ofentire wt CRISPR2 and flanking DNA sequence, approach used for complementation of CRISPR2 mutant with Wt CRISPR2. (C) PCR fragments generated to cleanly replace the entire sequence of one spacer with another, fragments diagramed were used in the replacement of spacer 17 with a spacer from PA2192. (D) Fragments amplified for deletion of spacers 2-21. PCR fragment 1 primers specifically amply the downstream homology region, and replace the spacer 21 position with PA2192 spacer sequence. PCR fragment 2 amplifies upstream homology region with PA2192 spacer sequence added at the terminus. (E) DNA fragments amplified to make point mutations within spacer 1. Primers with point mutations in spacer 1 were used in conjunction with the external primers to amplify upstream and downstream regions, overlapping in their now mutant spacer 1 sequence.
Supplemental Figure S2. Alignment of Cas2 and Cas3 N-terminal sequences. (A) Alignment of the N-terminus of Yersinia subtype Cas3 proteins from P. aeruginosa UCBPP-PA14 and E. coli UTI89 with previously published Cas2 sequences (Beloglazova et. al.) using CLUSTAL (SSO1404 and 8090=Sulfolobus solfataricusCas2, TT1823=Thermus thermophilusCas2, PF1117= Pyrococcus furiosusCas2, AF1876=Archaeoglobus fulgidusCas2, TM1796=Thermotoga maritime Cas2, NE0845=Nitrosomonas europaeaCas2). Boxed and highlighted residues were shown by Beloglazova et al. to be well conserved in Cas2 proteins. Arrows denote Cas2 residues shown to be involved in Cas2 nuclease activity (Beloglazova et. al.). (B) CLUSTAL derived N-J Tree with branch length, demonstrating the distinctive clustering of previously published Cas2 proteins from the Yersinia subtype Cas3 proteins.
Supplemental Table S1. Bacterial strains, plasmids and primers utilized in this study. Information for bacterial strains, plasmids, and primers used in this study are listed. PCR primer regions of homology utilized in S. cerevisiae plasmid recombination are denoted in bold.
Supplemental Table S2. Regions of bacteriophage DMS3 genome harboring high identity to CRISPR2 spacer 1. The gene or intergenic region of bacteriophage DMS3 genome with identity to PA14 CRISPR2 spacer 1 is shown in left column. 32 nucleotide sequence of DMS3 genomic locus harboring identity displayed in center column, with nucleotide positions that are not identical to spacer 1 sequence shaded grey. Level of identity between bacteriophage region and spacer 1 sequence are shown to the right. CRISPR2 spacer 1 sequence is shown at top for direct comparison.
Supplemental Table S1.
Strain, plasmid, or primer / Relevant characteristic / Reference or sourceS.cerevisiae INVSc1 / MATa his3D1 leu2 trp1-289 ura3-52 MAT his3D1 leu2 trp1-289 ura3-52 / Shanks et. al.
E.coli Top10 / F- mcrA (mrr-hsdRMS-mcrBC) f80lacZM15 lacX74recA1ara139(araleu)7697galUgalKrpsL (StrR) endA1nupG / Invitrogen
E.coli S17-1(pir) / thi pro hsdR- hsdM+ recA RP4-2::TcMu-Km::Tn7 / Shanks et. al.
SMC232 / Wild type PA14 / Zegans et. al.
SMC3884 / Wild type DMS3 Lysogen / Zegans et. al.
SMC3893 / PA14 PA14_CRISPR1 / Zegans et. al.
SMC4274 / PA14 PA14_CRISPR1 DMS3 lysogen / Zegans et. al.
SMC3894 / PA14 csy4 / Zegans et. al.
SMC4275 / PA14 csy4 DMS3 lysogen / Zegans et. al.
SMC4398 / PA14 csy4 +pMQ70 / This study
SMC4400 / PA14 csy4 +pMQ70-csy4 / This study
SMC4399 / PA14 csy4 DMS3 lysogen +pMQ70 / This study
SMC4401 / PA14 csy4 DMS3 lysogen +pMQ70-csy4 -pentaHis / This study
SMC4402 / PA14 csy4 +pMQ70-csy4 H29K-pentaHis / This study
SMC4403 / PA14 csy4 DMS3 lysogen +pMQ70-csy4 H29K-pentaHis / This study
SMC4446 / PA14 csy4 +pMQ70-csy4 H29A-pentaHis / This study
SMC4450 / PA14 csy4 DMS3 lysogen +pMQ70-csy4 H29A-pentaHis / This study
SMC4447 / PA14 csy4 +pMQ70- E. coli csy4-pentaHis / This study
SMC4451 / PA14 csy4 DMS3 lysogen +pMQ70- E. coli csy4-pentaHis / This study
SMC4449 / PA14 csy4 +pMQ70- E. coli csy4 H29A-pentaHis / This study
SMC4453 / PA14 csy4 DMS3 lysogen +pMQ70- E. coli csy4 H29A-pentaHis / This study
SMC4262 / PA14 csy3 / This study
SMC4263 / PA14 csy3 DMS3 lysogen / This study
SMC4264 / PA14 csy3+pMQ70 / This study
SMC4265 / PA14 csy3+pMQ70- csy3-pentaHis / This study
SMC4266 / PA14 csy3 DMS3 lysogen +pMQ70 / This study
SMC4267 / PA14 csy3 DMS3 lysogen +pMQ70- csy3-pentaHis / This study
SMC4250 / PA14 csy2 / This study
SMC4251 / PA14 csy2 DMS3 lysogen / This study
SMC4252 / PA14 csy2+pMQ70 / This study
SMC4253 / PA14 csy2+pMQ70-csy2-pentaHis / This study
SMC4253 / PA14 csy2 DMS3 lysogen +pMQ70 / This study
SMC4255 / PA14 csy2 DMS3 lysogen +pMQ70-csy2-pentaHis / This study
SMC4256 / PA14 csy1 / This study
SMC4257 / PA14 csy1 DMS3 lysogen / This study
SMC4258 / PA14 csy1+pMQ70 / This study
SMC4259 / PA14 csy1+pMQ70- csy1-pentaHis / This study
SMC4260 / PA14 csy1 DMS3 lysogen +pMQ70 / This study
SMC4261 / PA14 csy1 DMS3 lysogen +pMQ70- csy1-pentaHis / This study
SMC4268 / PA14 cas3 / This study
SMC4269 / PA14 cas3 DMS3 lysogen / This study
SMC4270 / PA14 cas3+pMQ70 / This study
SMC4271 / PA14 cas3+pMQ70-C-terminal pentaHis tagged cas3 / This study
SMC4533 / PA14 cas3+pMQ70-N-terminal pentaHis tagged cas3 / This study
SMC4272 / PA14 cas3 DMS3 lysogen +pMQ70 / This study
SMC4273 / PA14 cas3 DMS3 lysogen +pMQ70- C-terminal pentaHis cas3 / This study
SMC4534 / PA14 cas3 DMS3 lysogen +pMQ70- N-terminal pentaHis cas3 / This study
SMC4519 / PA14 cas3 DMS3 lysogen +pMQ70-cas3 / This study
SMC4650 / PA14cas3 + cas3 (chromosomal) / This study
SMC4651 / PA14cas3 + cas3 DMS3 lysogen (chromosomal) / This study
SMC4541 / PA14cas3 + cas3 D124A (chromosomal) / This study
SMC4542 / PA14cas3 + cas3 D124A DMS3 lysgoen (chromosomal) / This study
SMC4658 / PA14cas3 + cas3 D124A DMS3 lysgoen (chromosomal) +pMQ70-cas3 / This study
SMC4659 / PA14cas3 + cas3 D124A DMS3 lysgoen (chromosomal)
+pMQ70-cas3 D124A / This study
SMC4652 / PA14cas3 + cas3 D576A (chromosomal) / This study
SMC4653 / PA14cas3 + cas3 D576A DMS3 lysogen (chromosomal) / This study
SMC4661 / PA14cas3 + cas3 D576A DMS3 lysogen (chromosomal) +pMQ70-cas3 / This study
SMC4663 / PA14cas3 + cas3 D576A DMS3 lysogen (chromosomal)
+pMQ70-cas3 D576A / This study
SMC4277 / PA14 cas1 / This study
SMC4278 / PA14 cas1 DMS3 lysogen / This study
SMC3895 / PA14 PA14_CRISPR2 / Zegans et. al.
SMC4276 / PA14 PA14_CRISPR2 DMS3 lysogen / Zegans et. al.
SMC4646 / PA14 PA14_CRISPR2 +CRISPR2 / This study
SMC4647 / PA14 PA14_CRISPR2 DMS3 +CRISPR2 lysogen / This study
SMC4279 / PA14 PA14_CRISPR1-2 (CR) / This study
SMC4280 / PA14 PA14_CR DMS3 lysogen / This study
SMC4576 / PA14 PA14_CRISPR2 +CRISPR2 replacement of spacer20 / This study
SMC4540 / PA14 PA14_CRISPR2 +CRISPR2 replacement of spacer20 DMS3 lysogen / This study
SMC4578 / PA14 PA14_CRISPR2 +CRISPR2 replacement of spacer17 / This study
SMC4579 / PA14 PA14_CRISPR2 +CRISPR2 replacement of spacer17 DMS3 lysogen / This study
SMC4537 / PA14 PA14_CRISPR2 +CRISPR2 spacer7-21 / This study
SMC4538 / PA14 PA14_CRISPR2 +CRISPR2 spacer7-21 lysogen / This study
SMC4535 / PA14 PA14_CRISPR2 +CRISPR2 spacer1-21 / This study
SMC4536 / PA14 PA14_CRISPR2 +CRISPR2 spacer1-21 lysogen / This study
SMC4577 / PA14 PA14_CRISPR2 +CRISPR2 spacer2-21 / This study
SMC4539 / PA14 PA14_CRISPR2 +CRISPR2 spacer2-21 lysogen / This study
SMC4707 / PA14 PA14_CRISPR2 +CRISPR2 spacer1-2 / This study
SMC4737 / PA14 PA14_CRISPR2 +CRISPR2 spacer1-2 lysogen / This study
SMC4715 / PA14 PA14_CRISPR2 +CRISPR2 spacer1 SalI mutation / This study
SMC4716 / PA14 PA14_CRISPR2 +CRISPR2 spacer1 SalI mutation lysogen / This study
SMC4711 / PA14 PA14_CRISPR2 +CRISPR2 spacer1 BamHI mutation / This study
SMC4712 / PA14 PA14_CRISPR2 +CRISPR2 spacer1 BamHI mutation lysogen / This study
SMC4893 / DMS3-42 lysogen / This study
SMC4894 / DMS3-42 lysogen + DMS3-42 / This study
SMC4895 / DMS3-42 lysogen+ DMS3-42 T2G mutant / This study
SMC4896 / DMS3-42 lysogen+ DMS3-42 C234T mutant / This study
SMC4897 / DMS3-42 lysogen+ DMS3-42 G235T mutant / This study
SMC4898 / DMS3-42 lysogen+ DMS3-42 C238T mutant / This study
SMC4899 / DMS3-42 lysogen+ DMS3-42 C238G mutant / This study
SMC4900 / DMS3-42 lysogen+ DMS3-42 C243T mutant / This study
SMC4901 / DMS3-42 lysogen+ DMS3-42 C246T mutant / This study
SMC4902 / DMS3-42 lysogen+ DMS3-42 C249T mutant / This study
SMC4903 / DMS3-42 lysogen+ DMS3-42 C252T mutant / This study
SMC4904 / DMS3-42 lysogen+ DMS3-42 T254C mutant / This study
SMC4905 / DMS3-42 lysogen+ DMS3-42 C256A/C258A double mut. / This study
SMC4906 / DMS3-42 lysogen+ DMS3-42 G261T mutant / This study
SMC4907 / DMS3-42 lysogen+ DMS3-42 A262T mutant / This study
SMC4908 / DMS3-42 lysogen+ DMS3-42 C663T mutant / This study
SMC4909 / DMS3-42 lysogen+ DMS3-42 G681T mutant / This study
SMC4910 / CRISPR2 spacer 1 C7G salI DMS3-42 C238G lysogen / This study
Plasmids
pMQ70 / Arabinose-inducible expression vector; Cbr Apr URA3 / Shanks et. al.
pMQ30 / P. aeruginosa suicide vector for clean deletions; Gmr / Shanks et. al.
pMQ30-csy3KO / PA14_csy3 in-frame deletion plasmid; Gmr / This study
pMQ30-csy2KO / PA14_csy2 in-frame deletion plasmid; Gmr / This study
pMQ30-csy1KO / PA14_csy1 in-frame deletion plasmid; Gmr / This study
pMQ30-cas3KO / PA14_cas3 in-frame deletion plasmid; Gmr / This study
pMQ30-cas1KO / PA14_cas1 in-frame deletion plasmid; Gmr / This study
pMQ30-CRKO / PA14_CRISPR region deletion pasmid; Gmr / This study
pMQ70-csy4His / PA14_csy4 Complementation construct; Cbr Apr / This study
pMQ70-E. coli csy4His / E. coli UTI89_csy4 complementation construct; Cbr Apr / This study
pMQ70-csy4His (H29A) / PA14_csy4 complementation construct with catalytic residue H29A mutation; Cbr Apr / This study
pMQ70-csy4His (H29K) / PA14_csy4 complementation construct with catalytic residue H29K mutation; Cbr Apr / This study
pMQ70-E. coli csy4His (H29A) / E. coli UTI89_csy4 complementation construct with catalytic residue H29A mutation; Cbr Apr / This study
pMQ70-csy3His / PA14_csy3 complementation construct with C-terminal penta His tag; Cbr Apr / This study
pMQ70-Hiscsy2 / PA14_csy2 complementation construct with N-terminal penta His tag; Cbr Apr / This study
pMQ70-csy1His / PA14_csy1 complementation construct with C-terminal penta His tag; Cbr Apr / This study
pMQ70-cas3 / PA14_cas3 complementation construct; Cbr Apr / This study
pMQ70-cas3 (D124A) / PA14_cas3 complementation construct with catalytic residue D124A mutation; Cbr Apr / This study
pMQ70-cas3 (D576A) / PA14_cas3 complementation construct with catalytic residue D576A mutation; Cbr Apr / This study
pMQ70-Hiscas3 / PA14_cas3 complementation construct with N-terminal penta His tag; Cbr Apr / This study
pMQ70-cas3His / PA14_cas3 complementation construct with C-terminal penta His tag; Cbr Apr / This study
pMQ30-cas3 KON / cas3 allelic replacement construct; Gmr / This study
pMQ30-cas3 KON (D124A) / cas3 allelic replacement construct with catalytic D124A mutation; Gmr / This study
pMQ30-cas3 KON (D576A) / cas3 allelic replacement construct with catalytic D576A mutation; Gmr / This study
pMQ30-CRISPR2 KON / CRISPR2 allelic replacement construct; Gmr / This study
pMQ30-CRISPR2 spacer 20 KON / CRISPR2 allelic replacement construct with PA2192 spacer replacing PA14 CRISPR2 spacer 20; Gmr / This study
pMQ30-CRISPR2 spacer 17 KON / CRISPR2 allelic replacement construct with PA2192 spacer replacing PA14 CRISPR2 spacer 17; Gmr / This study
pMQ30-CRISPR2 spacers 7-21KON / CRISPR2 allelic replacement construct with PA2192 spacer replacing PA14 CRISPR2 spacers 7-21; Gmr / This study
pMQ30-CRISPR2 spacers 2-21KON / CRISPR2 allelic replacement construct with PA2192 spacer replacing PA14 CRISPR2 spacers 2-21; Gmr / This study
pMQ30-CRISPR2 spacers 1-21 KON / CRISPR2 allelic replacement construct with PA2192 spacer replacing PA14 CRISPR2 spacers 1-21; Gmr / This study
pMQ30-CRISPR2 spacers 1-2 KON / CRISPR2 allelic replacement construct with PA2192 spacer replacing PA14 CRISPR2 spacers 1-2; Gmr / This study
pMQ30-CRISPR2 spacer1 SalI mutation KON / CRISPR2 allelic replacement construct with SalI site added to CRISPR2 spacer 1; Gmr / This study
pMQ30-CRISPR2 spacer1 BamHI mutation KON / CRISPR2 allelic replacement construct with BamHI site added to CRISPR2 spacer 1; Gmr / This study
pMQ70-DMS3-42 / DMS3-42 expression construct with C-terminal penta His tag; Cbr Apr / This study
pMQ30-DMS3-42KO / DMS3-42 in-frame deletion plasmid; Gmr / This study
pMQ30-DMS3-42KON / DMS3-42 allelic replacement construct; Gmr / This study
pMQ30-DMS3-42 T2G mutation KON / DMS3-42allelic replacement construct with translational start site mutation T2G; Gmr / This study
pMQ30-DMS3-42 C234T mutation KON / DMS3-42allelic replacement construct with point mutation C234T; Gmr / This study
pMQ30-DMS3-42 G235T mutation KON / DMS3-42allelic replacement construct with point mutation G235T. Results in DMS3-42 A79S substitution ; Gmr / This study
pMQ30-DMS3-42 C238T mutation KON / DMS3-42allelic replacement construct with point mutation C238T ; Gmr / This study
pMQ30-DMS3-42 C238G mutation KON / DMS3-42allelic replacement construct with point mutation C238G. Resulted in L80V substitution ; Gmr / This study
pMQ30-DMS3-42 C243T mutation KON / DMS3-42allelic replacement construct with point mutation C243T ; Gmr / This study
pMQ30-DMS3-42 C246T mutation KON / DMS3-42 allelic replacement construct with point mutation C246T ; Gmr / This study
pMQ30-DMS3-42 C249T mutation KON / DMS3-42 allelic replacement construct with point mutation C249T ; Gmr / This study
pMQ30-DMS3-42 C252T mutation KON / DMS3-42 allelic replacement construct with point mutation C252T ; Gmr / This study
pMQ30-DMS3-42 T254C mutation KON / DMS3-42 allelic replacement construct with point mutation T254C. Results in L85P substitution ; Gmr / This study
pMQ30-DMS3-42 C256A and C258A double mutation KON / DMS3-42 allelic replacement construct with double mutation C256A and C258A ; Gmr / This study
pMQ30-DMS3-42 G261T mutation KON / DMS3-42 allelic replacement construct with point mutation G261T ; Gmr / This study
pMQ30-DMS3-42 A262T mutation KON / DMS3-42 allelic replacement construct with point mutation A262T. Results in M88L substitution; Gmr / This study
pMQ30-DMS3-42 C663T mutation KON / DMS3-42 allelic replacement construct with point mutation C663T ; Gmr / This study
pMQ30-DMS3-42 G681T mutation KON / DMS3-42 allelic replacement construct with point mutation G681T; Gmr / This study
Primers
33310KO primer 1 (60) / cgcttctgcgttctgatttaatctgtatcaggctgaTCGAAGATGAGCGTGACTGATCC
33310KO primer 2(61) / GCGAATGTCGAGGTAGTGGTCCATGGCTTGCTGATTTCCTTATGCGATGGCG
33310KO primer 3(64) / CGCCATCGCATAAGGAAATCAGCAAGCCATGGACCACTACCTCGACATTCGC
33310KO primer 4(62) / GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGGAAAAGAACATGGCCCGGCTCCCG
33320KO primer 1(64) / ccgcttctgcgttctgatttaatctgtatcaggctgaAGGCTGGTCAGCGATGTGGTGGGC
33320KO primer 2(63) / CTCAGTATTGGCTTGGACATGCTGATTTCCTTAATCAGTCACGCTCATCTTCGAGTATCTCC
33320KO primer 3(64) / GGAGATACTCGAAGATGAGCGTGACTGATTAAGGAAATCAGCATGTCCAAGCCAATACTGAG
33320KO primer 4(63) / GAGCGGATAACAATTTCACACAGGAAACAGCTAGGTTGGCGATCACATAATGCTGTTGCTCC
33330KO primer 1(62) / taatctgtatcaggctgaaaatcttctctcatccgccaaaCAGTATTGGCTTGGACATGCTGATTTCCTT
33330KO primer 5(63) / GTCAATGACAATCGACCAAGGAGGCATGGATGAGCGTGACTGATCCCGAGGCG
33330KO primer 6(63) / CGCCTCGGGATCAGTCACGCTCATCCATGCCTCCTTGGTCGATTGTCATTGAC
33330KO primer 4(61) / GTGAGCGGATAACAATTTCACACAGGAAACAGCTTGTGTGTCTACCATTCGCGGCATCC
33340KO primer 1(62) / tcaggctgaaaatcttctctcatccgccaaaacagccaagTTGTTATGGACTGTCTTGGCAAGAAAACGC
33340KO primer 5(63) / AGCGGAGCACGGTGTCCGCATGAGGAGCACAGCTTTCGAATGCGAGC
33340KO primer 6(63) / GCTCGCATTCGAAAGCTGTGCTCCTCATGCGGACACCGTGCTCCGCT
33340KO primer 4(62) / TGTGAGCGGATAACAATTTCACACAGGAAACAGCTAGATCCGATAGGGACAGGCCACGG
33350KO primer 1(63) / ccgcttctgcgttctgatttaatctgtatcaggctgaAAGCCGGGCCGATGTTCGAGCC
33350KO primer 2(62) / GCATTGCGACACCAGCAGGATGTTCATGTTGACCGTGGCCTGTCCCTATCGG
33350KO primer 3(63) / CCGATAGGGACAGGCCACGGTCAACATGAACATCCTGCTGGTGTCGCAATGC
33350KO primer 4(63) / GAGCGGATAACAATTTCACACAGGAAACAGCTATGTGCAAGGGGAAGTTCGGGTCGCCC
CRKO primer 1(63) / caaattctgttttatcagaccgcttctgcgttctgatCTAGAGCGGGCGCAACTTGCTC
CRKO primer 2(62) / GACGATTTCTTACGCCCTTATAAATCAGCAAGCTGTGTTCTCCATGCCGGCGTAATG
CRKO primer 3(62) / CATTACGCCGGCATGGAGAACACAGCTTGCTGATTTATAAGGGCGTAAGAAATCGTC
CRKO primer 4 / Same primer as CRISPR2 KO4
csy4CC Primer1(63) / aaatcttctctcatccgccTCAatgatgatgatgatgatgGAACCAGGGAACGAAACCTCCTTTGCTCAAC
csy4CC Primer2(63) / CCATACCCGTTTTTTTGGGCTAGCCCAAGGAAGCACAACCATGGACCACTACCTCGACATTCGCTTGC
csy4 H29A 1 / TGTGCCACCAGGGCCTGGGCGA
csy4 H29A 2 / TCGCCCAGGCCCTGGTGGCACA
E.colicsy4 CC 1(63) / aaatcttctctcatccgccTCAatgatgatgatgatgatgAAACCAGGGAATAGTGGCGGTTGCGC
E.colicsy4 CC 2(63) / TACCCGTTTTTTTGGGCTAGCCCAAGGAAGCACAACCATGGATCACTACCTTGAGATCCGCGTTCT
csy4 H29K 1 / TGTCCACCAGGGCCTGTTTGAGCTT
csy4 H29K 2 / AAGCTCAAACAGGCCCTGGTGGCAC
csy3CC Primer1(64) / aaatcttctctcatccgccTCAatgatgatgatgatgatgCTTCTCTTCGGCTTCACCGAACACGC
csy3CC Primer2(63) / CCATACCCGTTTTTTTGGGCTAGCCCAAGGAAGCACAACCATGTCCAAGCCAATACTGAGCACCGCT
csy2CC Primer1(63) / tgtatcaggctgaaaatcttctctcatccgccTCATGCGATGGCGTGTTCGACGAAACG
csy2CC Primer2(64) / TACCCGTTTTTTTGGGCTAGCCCAAGGAAGCACAACCATGCATCATCATCATCATCATATGAGCGTGACTGATCCCGAGGCG
csy1CC Primer1(63) / aaatcttctctcatccgccTCAatgatgatgatgatgatgGTCACGCTCATCTTCGAGTATCTCCTTGA
csy1CC Primer2(64) / CCATACCCGTTTTTTTGGGCTAGCCCAAGGAAGCACAACCATGACCTCTCCCCTCCCAACGCCTACG
cas3 KON For(70)- / tcaggctgaaaatcttctctcatccgccaaaacagccaagTTGTTATGGACTGTCTTGGCAAGAAAACGCTGCAGGGTACG
cas3 KON rev(70)- / TGTGAGCGGATAACAATTTCACACAGGAAACAGCTAGATCCGATAGGGACAGGCCACGGTCAACATGGACGACATTTC
Cas3 CC1 (70)- / taatctgtatcaggctgaaaatcttctctcatccgccTCAGTTGTATTTCTTGAACCCCAAATAAGGGTGGTAGCTCCATCCCTGG
Cas3 CC4(70)- / CCATACCCGTTTTTTTGGGCTAGCCCAAGGAAGCACAACCATGAACATCCTGCTGGTGTCGCAATGCGAAAAGCG
Cas3 D124A 2(70)- / GATGGCAGCGCTGTTCCACGCTATCGGCAA
Cas3 D124A 3(70)- / TTGCCGATAGCGTGGAACAGCGCTGCCATC
Cas3 D576A 2(71)- / ACTTCCGACCTGGTGCTCGCCGAGGTCGACGAC
Cas3 D576A 3(71)- / GTCGTCGACCTCGGCGAGCACCAGGTCGGAAGT
N-termHis Cas3 CC4(70)- / ATACCCGTTTTTTTGGGCTAGCCCAAGGAAGCACAACCATGCATCATCATCATCATCATAACATCCTGCTGGTGTCGCAATGCGAAAAGC
CRISPR2 KON Primer1(71) / agggttttcccagtcacgacgttgtaaaacgacggccGTAGCACCGCCAGTCCATGGGGAATGCCCAGCACCC
CRISPR2 KON Primer2(71) / TGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGCCCATGGCTAAGCCGAATGATACGTCCAACCCCGAGGTAACAG
CRISPR2 20 1(73) / AAGATGGTCGACGTGCTGATCCTGGCAATGCGGTTCACTGCCGTGTAGGCAGCTAAGAAATTGCCGAGTACGATGC
CRISPR2 20 2(74) / cgcattgccaggatcagcacgtcgaccatcttTTTCTTAGCTGCCTACACGGCAGTGAACGAGAGCCGCAGGG
CRISPR2 17 1(74) / cgcattgccaggatcagcacgtcgaccatcttTTTCTTAGCTGCCTACACGGCAGTGAACCGTCTCGATGCTGCC
CRISPR2 17 2(74) / AAGATGGTCGACGTGCTGATCCTGGCAATGCGGTTCACTGCCGTGTAGGCAGCTAAGAAAATCCTCGAACAGCCCGGGTTC
CRISPR2 7-21 1(71) / AAGATGGTCGACGTGCTGATCCTGGCAATGCGGTTCACTGCCGTGTAGGCAGCTAAGAAACTCGAACCCACCTC
CRISPR2 7-21 2(71) / cgcattgccaggatcagcacgtcgaccatcttTTTCTTAGCTGCCTACACGGCAGTGAACGCGCTGAC
CRISPR2 2-21 1(71) / AAGATGGTCGACGTGCTGATCCTGGCAATGCGGTTCACTGCCGTGTAGGCAGCTAAGAAACTCGAACCCACCTC
CRISPR2 1-21 1(71) / AAGATGGTCGACGTGCTGATCCTGGCAATGCGGTTCACTGCCGTGTAGGCAGCTAAGAAACTCGAACCCACCTC
CRISPR2 1-21 2(71) / cgcattgccaggatcagcacgtcgaccatcttTTTCTTAGCTGCCTACACGGCAGTGAACTAGCTCCGAAAACCTATAAC
CRISPR2 1-2 1(72) / AAGATGGTCGACGTGCTGATCCTGGCAATGCGGTTCACTGCCGTGTAGGCAGCTAAGAAAGACCTGGTTTTGGCCG
CRISPR2 1-2 2(71) / cgcattgccaggatcagcacgtcgaccatcttTTTCTTAGCTGCCTACACGGCAGTGAACTAGCTCCGAAAACCTATAAC
CRISPR2 spacer1 SalI 1 (70) / CACCGCGGTCGACTACTACAACGTCCGGCTGAT
CRISPR2 spacer1 SalI 2 (70) / ATCAGCCGGACGTTGTAGTAGTCGACCGCGGTG
CRISPR2 spacer1 BamHI 1 (70) / CACCGCGCTCGACTACTACAAGGATCCGGCTGAT
CRISPR2 spacer1 BamHI 2 (70) / ATCAGCCGGATCCTTGTAGTAGTCGAGCGCGGTG
DMS3-42 1 (68)- / taatctgtatcaggctgaaaatcttctctcatccgccaaaCTACGAGAAGCTCCAGCAGATTCGGCCGATC
DMS3-42 2 (68)- / CTGTGAAGTAAGCCATTTCAGAATTCCTAGTTTCCGAGTATT TGCTTGACGGCCTGGC
DMS3-42 3 (68)- / TACTCGGAAACTAGGAATTCTGAAATGGCTTACTTCACAGGAACAGCGAACAACCCG
DMS3-42 4 (68)- / GTGAGCGGATAACAATTTCACACAGGAAACAGCTCGATATTCGGCGTGATGCCGGCAACGATCCC
DMS3-42 C238T for(68)- / CTACGGCGATACCGCCTTGGACTACTACAACCTTCG
DMS3-42 C238T Rev(68)- / CGAAGGTTGTAGTAGTCCAAGGCGGTATCGCCGTAG
DMS3-42 C252T for(68)- / GCCCTGGACTACTACAATCTTCGCCTGATGGGC
DMS3-42 C252T Rev(68)- / GCCCATCAGGCGAAGATTGTAGTAGTCCAGGGC
42 T2G For (68)- / GGAAACTAGGAATTCAGGGCTACTGAAATCGGCACCG
42 T2G Rev (68)- / CGGTGCCGATTTCAGTAGCCCTGAATTCCTAGTTTCC
C234TFor(68) / GAGCTACGGCGATACTGCCCTGGACTACTACAACC
C234T Rev(68) / GGTTGTAGTAGTCCAGGGCAGTATCGCCGTAGCTC
C246T For(68) / GATACCGCCCTGGACTATTACAACCTTCGCCTGATGGG
C246T Rev(68) / CCCATCAGGCGAAGGTTGTAATAGTCCAGGGCGGTATC
C663T For(68) / GCCGTTCAAGCAACGAGTCCGATTACAACAATCGG
C663TRev(68) / CCGATTGTTGTAATCGGACTCGTTGCTTGAACGGC
G681T For(68) / CAACAATCGGACCCTTCTGCCGCTTGGCTG
G681T Rev(68) / CAGCCAAGCGGCAGAAGGGTCCGATTGTTG
C243T F(68)- / GGCGATACCGCCCTGGATTACTACAACCTTCGCC
C243T R(68)- / GGCGAAGGTTGTAGTAATCCAGGGCGGTATCGCC
C249T F(68)- / CGATACCGCCCTGGACTACTATAACCTTCGCCTG
C249T R(68)- / CAGGCGAAGGTTATAGTAGTCCAGGGCGGTATCG
G261T F(68)- / CTACAACCTTCGCCTTATGGGCGGCACGGC
G261T R(68)- / GCCGTGCCGCCCATAAGGCGAAGGTTGTAG
C238G F(68)- / CTACGGCGATACCGCCGTGGACTACTACAACCTTC
C238G R(68)- / GAAGGTTGTAGTAGTCCACGGCGGTATCGCCGTAG
G235T F(68)- / GAGCTACGGCGATACCTCCCTGGACTACTACAACCTTC
G235T R(68)- / GAAGGTTGTAGTAGTCCAGGGAGGTATCGCCGTAGCTC
T254C F(68)- / CCCTGGACTACTACAACCCTCGCCTGATGGGC
T254C R(68)- / GCCCATCAGGCGAGGGTTGTAGTAGTCCAGGG
C256A/C258A F(68)- / GGACTACTACAACCTTAGACTGATGGGCGGCACGG
C256A/C258A R(68)- / CCGTGCCGCCCATCAGTCTAAGGTTGTAGTAGTCC
A262T F(68)- / CTACAACCTTCGCCTGTTGGGCGGCACGG
A262T R(68)- / CCGTGCCGCCCAACAGGCGAAGGTTGTAG
DMS3-42 check F(72)- / CAACGAAATGGACGCCCGGCAGCGCATCCTGGACATTC
DMS3-42 check R(72)- / CGACCGCTGCCAGGCTGATGTCCTGGTTCAGCTC
DMS3-42 seq For(65)- / CCCGATTCTGGCATCCAACGACGAGTTTATGAC
Region harboring identity / Sequence / Identity
CRISPR2 spacer 1 / ACCGCGCTCGACTACTACAACGTCCGGCTGAT
DMS3-42 / ACCGCCCTGGACTACTACAACCTTCGCCTGAT / 27/32
DMS3-42 / AACGAGTCCGACTACAACAATCGGACCCTGCT / 19/32
DMS3-4 / CAGCAGATTGCCGAATACAACGACCGGCCGCA / 18/32
Promoter DMS3-1/2 / GAAATCCCCCGCGACAACAACGTCCCGCTGGG / 18/32
DMS3-44 / CTGGAAGAGAACTACTACAACCGCCTGTGGCT / 17/32
DMS3-18 / GAGCTGCTCGACTACCACCAGCGTGGCCGGCA / 16/32
DMS3-1 / CCAAACTGTCCCACAGCGTCTGTCCGGCTGGA / 11/32
Supplemental Table S2.