Supporting InformationSemenova et al. 10.1073/pnas.1104144108
Fig. S1. Design of nontargeting CRISPR (clustered regularly interspaced short palindromic repeats) (pWUR477, S1) and M13 g8-targeting CRISPR cassettes;rectangles indicate spacers, rhombi—repeats. Below synthetic DNA used for generating M13 phage matching CRISPR cassette plasmid is shown. Two oligo-nucleotides were annealed to create double-stranded DNAwith sticky EcoRI and BamHI ends. The g8 spacer is shown in red color, repeat sequences are shownin lowercase.
Fig. S2. (A) Natural M13 escape mutants obtained on lawns of cells expressing g8 crRNA. The wild-type sequence is shown (Top). Individual mutations areindicated by red-color font. Numbers indicate frequencies of individual mutations (a total of 50 escape mutant phages was sequenced). (B) Engineered pointmutations in g8 protospacer, which were not included in Fig. 1B. Mutations indicated with red color led to escape, whereas mutations shown in black wererestricted by CRISPR/Cas (the phage mutants had nonescape phenotype on lawns of cells expressing the g8 crRNA). PAM, protospacer-adjacent motif.
Semenova et al. www.pnas.org/cgi/doi/10.1073/pnas.1104144108 1 of 10
Fig. S3. Mutations in PAM and protospacer seed region abolish CRISPR/Cas mediated plasmid transformation inhibition. (A) Transformation efficiencies ofpUC19, pWUR610, and pWUR610 escape mutant series. pWUR610 is a pUC19 plasmid containing the J3 protospacer on a 350-bp fragment of bacteriophage λgenome (Table S1). The recA− strain Escherichia coli KRX (Promega) was used to overproduce Cascade, Cas3, and J3 crRNA. Error bars indicate the standarddeviation. (B) Overview of the identified escape mutants. The sequence of the protospacer as well as escape point mutations are shown.
Fig. S4. Escape mutants obtained on lawns of cells expressing g8 crRNA with individual nonescape phages carrying point mutations in the g8 protospacer.Each row indicates a nonrestricted point mutant of M13, with mutational substitution indicated with black font color. In red, substitutions observed in phagesthat escaped the block imposed by cells expressing the g8 crRNA are shown.
Semenova et al. www.pnas.org/cgi/doi/10.1073/pnas.1104144108 2 of 10
Fig. S5. Escape mutants obtained on lawns of cells expressing g8 crRNA with triple (A), quadruple (B), and quintuple (C) nonescape phages carrying pointmutations in the g8 protospacer. See Fig. S3 legend for details.
Fig. S6. Quadruple and quintuple mutants that escape CRISPR/Cas-imposed block on cells expressing g8 crRNA.
Semenova et al. www.pnas.org/cgi/doi/10.1073/pnas.1104144108 3 of 10
Fig. S7. Phenotypes of indicated phages on lawns of hns− E. coli cells containing an engineered g8 CRISPR spacer in their chromosomal CRISPR cassette. SeeFig. 1 legend for nomenclature.
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Probe pairPosition
(mutation)Phenotype Kd (nM) EMSA gel
BG3233+BG3234 wild type 24 ± 9
BG3333+BG3334 -4 (G C) nonescape 31 ± 7
BG3235+BG3236 -3 (A C) escape 285 ± 32
BG3237+BG3238 -2 (T C) escape > 1,230
BG3239+BG3240 -1 (G T) escape > 1,230
BG3241+BG3242 1 (C T) escape > 1,230
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BG3243+BG3244 2 (T A) escape 270 ± 20
BG3245+BG3246 3 (G T) escape 790 ± 254
BG3247+BG3248 4 (T G) escape > 1,230
BG3249+BG3250 5 (C G) escape 782 ± 132
BG3251+BG3252 6 (T A) nonescape 38 ± 14
BG3253+BG3254 7 (T C) escape > 1,230
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BG3255+BG3256 8 (T C) escape 1176 ± 112
BG3331+BG3332 9 (C T) nonescape 38 ± 11
BG3365+BG3366 10 (G T) nonescape 310 ± 28
BG3367+BG3368 15 (T A) nonescape 439 ± 43
BG3369+BG3370 24 (C T) nonescape 45 ± 20
BG3371+BG3372 30 (C A) nonescape 29 ± 8
Fig. S8. Electrophoretic mobility shift assays of Cascade complex binding various double-stranded DNA probes. The probes are listed in Table S2. Cascadeconcentration range (nM): 1200, 600, 300, 150, 75, 37.5, 18.8, 9.4, 4.7, 2.3, 1.2, respectively.
Semenova et al. www.pnas.org/cgi/doi/10.1073/pnas.1104144108 7 of 10
Table S1. Plasmids used in this study
Plasmids Description and order of genes (5′-3′) Restriction sites Primers Source
pWUR477 nontargeting CRISPR in pACYCDuet-1 Brouns et al. (1)pWUR397 cas3 in pRSF-1b, no tags Brouns et al. (1)pWUR399 casA-casB-casC-casD-casE-cas1-cas2 in pCDF-1b, no tags Brouns et al. (1)pWUR400 casA-casB-casC-casD-casE in pCDF-1b, no tags Brouns et al. (1)pWUR408 casA in pRSF-1b, no tags Brouns et al. (1)pWUR514 casB with Strep-tag II (N-term)-casC-casD-CasE in pET52b Jore et al. (2)pWUR610 lambda fragment 17918–18250, in pUC19 BamHI/HindIII BG3218 + BG3219 this studypWUR615 E. coli CRISPR, 7 x spacer g8, in pACYCDuet-1* EcoNI/Acc65I Geneart, GermanypWUR630 E. coli CRISPR, 4 x spacer J3, in pACYCDuet-1† NcoI/KpnI Geneart, Germany
*pWUR615—E. coli CRISPR, 7x spacer g8 (underlined), in pACYCDuet-1CCTGCATTAGGTAATACGACTCACTATAGGATAAACCGCTGTCTTTCGCTGCTGAGGGTGACGATCCCGCGAGTTCCCCGCGCCAGCGGGGATAAACCGCTGTCTTTCGCTGCTGAGGGTGACGATCCCGCGAGTTCCCCGCGCCAGCGGGGATAAACCGCTGTCTTTCGCTGCTGAGGGTGACGATCCCGCGAGTTCCCCGCGCCAGCGGGGATAAACCGCTGTCTTTCGCTGCTGAGGGTGACGATCCCGCGAGTTCCCCGCGCCAGCGGGGATAAACCGCTGTCTTTCGCTGCTGAGGGTGACGATCCCGCGAGTTCCCCGCGCCAGCGGGGATAAACCGCTGTCTTTCGCTGCTGAGGGTGACGATCCCGCGAGTTCCCCGCGCCAGCGGGGATAAACCGCTGTCTTTCGCTGCTGAGGGTGACGATCCCGCGAGTTCCCCGCGCCAGCGGGGATAAACCGGGTACC.†pWUR630—E. coli CRISPR, 4x spacer J3 (underlined), in pACYCDuet-1CCATGGAAACAAAGAATTAGCTGATCTTTAATAATAAGGAAATGTTACATTAAGGTTGGTGGGTTGTTTTTATGGGAAAAAATGCTTTAAGAACAAATGTATACTTCTAGAGAGTTCCCCGCGCCAGCGGGGATAAACCGCCAGTGATAAGTGGAATGCCATGTGGGCTGTCGAGTTCCCCGCGCCAGCGGGGATAAACCGCCAGTGATAAGTGGAATGCCATGTGGGCTGTCGAGTTCCCCGCGCCAGCGGGGATAAACCGCCAGTGATAAGTGGAATGCCATGTGGGCTGTCGAGTTCCCCGCGCCAGCGGGGATAAACCGCCAGTGATAAGTGGAATGCCATGTGGGCTGTCGAGTTCCCCGCGCCAGCGGGGATAAACCGCAGCTCCCATTTTCAAACCCAGGTACC.
1. Brouns SJ, et al. (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321:960–964.2. Jore MM, et al. (2011) Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat Struct Mol Biol 18:529–536.
Semenova et al. www.pnas.org/cgi/doi/10.1073/pnas.1104144108 8 of 10
Table
S2.Prim
ersusedin
this
study
Experim
ent
Prim
erSe
quen
ce(5′-3′)
Description
M13
mutagen
esis*
N-4
GCTA
CCCTC
GTT
CCHATG
CTG
TCTT
TCGC
Mutationat
position−4
N2
CCTC
GTT
CCGATG
CVGTC
TTTC
GCTG
CTG
Mutationat
position2
N4
TCGTT
CCGATG
CTG
VCTT
TCGCTG
CTG
AG
Mutationat
position4
N5
CGTT
CCGATG
CTG
TDTT
TCGCTG
CTG
AGG
Mutationat
position5
N6
GTT
CCGATG
CTG
TCVTT
CGCTG
CTG
AGGG
Mutationat
position6
N7
TTCCGATG
CTG
TCTV
TCGCTG
CTG
AGGGT
Mutationat
position7
N8
TCCGATG
CTG
TCTT
VCGCTG
CTG
AGGGTG
Mutationat
position8
N9
CCGATG
CTG
TCTT
TTGCTG
CTG
AGGGTG
AMutationat
position9
N10
CGATG
CTG
TCTT
TCHCTG
CTG
AGGGTG
AC
Mutationat
position10
N11
GATG
CTG
TCTT
TCGDTG
CTG
AGGGTG
ACG
Mutationat
position11
N12
ATG
CTG
TCTT
TCGCVGCTG
AGGGTG
ACGA
Mutationat
position12
N13
TGCTG
TCTT
TCGCTH
CTG
AGGGTG
ACGAT
Mutationat
position13
N14
GCTG
TCTT
TCGCTG
DTG
AGGGTG
ACGATC
Mutationat
position14
N15
CTG
TCTT
TCGCTG
CVGAGGGTG
ACGATC
CMutationat
position15
N16
TGTC
TTTC
GCTG
CTH
AGGGTG
ACGATC
CC
Mutationat
position16
N17
GTC
TTTC
GCTG
CTG
BGGGTG
ACGATC
CCG
Mutationat
position17
N18
TCTT
TCGCTG
CTG
AAGGTG
ACGATC
CCGC
Mutationat
position18
N19
CTT
TCGCTG
CTG
AGHGTG
ACGATC
CCGCA
Mutationat
position19
N22
TCGCTG
CTG
AGGGTH
ACGATC
CCGCAAAA
Mutationat
position22
N23
CGCTG
CTG
AGGGTG
BCGATC
CCGCAAAAG
Mutationat
position23
N24
GCTG
CTG
AGGGTG
ATG
ATC
CCGCAAAAGC
Mutationat
position24
N27
GCTG
AGGGTG
ACGACCCCGCAAAAGCGGC
Mutationat
position27
N30
GAGGGTG
ACGATC
CDGCAAAAGCGGCCTT
Mutationat
position30
N33
GGTG
ACGATC
CCGCBAAAGCGGCCTT
TAA
Mutationat
position32
N9-11
(T6C
)CCGATG
CTG
TCCTT
TNNTG
CTG
AGGGTG
ACG
Mutationat
positions6,
9–11
N18
-20(T6
C,C30
A)
TCCTT
CGCTG
CTG
AANNTG
ACGATC
CAGCAA
Mutationat
positions6,
18–2
0,30
N27
-29(C30
A)
GCTG
AGGGTG
ACGACNNAGCAAAAGCGGCCT
Mutationat
positions27
-30
N29
-32
CGCTG
CTG
AGGGTG
ACGATC
DDAGTA
AAGCGGCCTT
TAACTC
Mutationat
positions29
–32
N28
-32
CTT
TCGCTG
CTG
AGGGTG
ACGATD
DDAGTA
AAGCGGCCTT
TAACTC
Mutationat
positions28
–32
Mutantphag
esequen
cing
G8_
FCTT
TAGTC
CTC
AAAGCCTC
TGg8protospacer
forw
ardprimer
G8_
RGCTT
GCTT
TCGAGGTG
AATT
TCg8protospacer
reve
rseprimer
Plasmid
construction
BG32
18GGCCCGGATC
CGTC
GGGCGAGCGATG
ATG
CG
lambdafrag
men
t+Bam
HI(fw)
BG32
19CGCGCAAGCTT
CATC
GGCGTT
TCATT
CCCGTT
Tlambdafrag
men
t+HindIII
(rv)
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Experim
ent
Prim
erSe
quen
ce(5′-3′)
Description
EMSA
BG32
33GCTA
CCCTC
GTT
CCGATG
CTG
TCTT
TCGCTG
CTG
AGGGTG
ACGATC
CCGCAAAAGCGGCCTT
TAA
Nativetarget
BG32
34TT
AAAGGCCGCTT
TTGCGGGATC
GTC
ACCCTC
AGCAGCGAAAGACAGCATC
GGAACGAGGGTA
GC
Nativetarget
BG32
35GCTA
CCCTC
GTT
CCGCTG
CTG
TCTT
TCGCTG
CTG
AGGGTG
ACGATC
CCGCAAAAGCGGCCTT
TAA
Mutationat
position−3
BG32
36TT
AAAGGCCGCTT
TTGCGGGATC
GTC
ACCCTC
AGCAGCGAAAGACAGCAGCGGAACGAGGGTA
GC
Mutationat
position−3
BG32
37GCTA
CCCTC
GTT
CCGACGCTG
TCTT
TCGCTG
CTG
AGGGTG
ACGATC
CCGCAAAAGCGGCCTT
TAA
Mutationat
position−2
BG32
38TT
AAAGGCCGCTT
TTGCGGGATC
GTC
ACCCTC
AGCAGCGAAAGACAGCGTC
GGAACGAGGGTA
GC
Mutationat
position−2
BG32
39GCTA
CCCTC
GTT
CCGATT
CTG
TCTT
TCGCTG
CTG
AGGGTG
ACGATC
CCGCAAAAGCGGCCTT
TAA
Mutationat
position−1
BG32
40TT
AAAGGCCGCTT
TTGCGGGATC
GTC
ACCCTC
AGCAGCGAAAGACAGAATC
GGAACGAGGGTA
GC
Mutationat
position−1
BG32
41GCTA
CCCTC
GTT
CCGATG
TTGTC
TTTC
GCTG
CTG
AGGGTG
ACGATC
CCGCAAAAGCGGCCTT
TAA
Mutationat
position1
BG32
42TT
AAAGGCCGCTT
TTGCGGGATC
GTC
ACCCTC
AGCAGCGAAAGACAACATC
GGAACGAGGGTA
GC
Mutationat
position1
BG32
43GCTA
CCCTC
GTT
CCGATG
CAGTC
TTTC
GCTG
CTG
AGGGTG
ACGATC
CCGCAAAAGCGGCCTT
TAA
Mutationat
position2
BG32
44TT
AAAGGCCGCTT
TTGCGGGATC
GTC
ACCCTC
AGCAGCGAAAGACTG
CATC
GGAACGAGGGTA
GC
Mutationat
position2
BG32
45GCTA
CCCTC
GTT
CCGATG
CTT
TCTT
TCGCTG
CTG
AGGGTG
ACGATC
CCGCAAAAGCGGCCTT
TAA
Mutationat
position3
BG32
46TT
AAAGGCCGCTT
TTGCGGGATC
GTC
ACCCTC
AGCAGCGAAAGAAAGCATC
GGAACGAGGGTA
GC
Mutationat
position3
BG32
47GCTA
CCCTC
GTT
CCGATG
CTG
GCTT
TCGCTG
CTG
AGGGTG
ACGATC
CCGCAAAAGCGGCCTT
TAA
Mutationat
position4
BG32
48TT
AAAGGCCGCTT
TTGCGGGATC
GTC
ACCCTC
AGCAGCGAAAGCCAGCATC
GGAACGAGGGTA
GC
Mutationat
position4
BG32
49GCTA
CCCTC
GTT
CCGATG
CTG
TGTT
TCGCTG
CTG
AGGGTG
ACGATC
CCGCAAAAGCGGCCTT
TAA
Mutationat
position5
BG32
50TT
AAAGGCCGCTT
TTGCGGGATC
GTC
ACCCTC
AGCAGCGAAACACAGCATC
GGAACGAGGGTA
GC
Mutationat
position5
BG32
51GCTA
CCCTC
GTT
CCGATG
CTG
TCATT
CGCTG
CTG
AGGGTG
ACGATC
CCGCAAAAGCGGCCTT
TAA
Mutationat
position6
BG32
52TT
AAAGGCCGCTT
TTGCGGGATC
GTC
ACCCTC
AGCAGCGAATG
ACAGCATC
GGAACGAGGGTA
GC
Mutationat
position6
BG32
53GCTA
CCCTC
GTT
CCGATG
CTG
TCTC
TCGCTG
CTG
AGGGTG
ACGATC
CCGCAAAAGCGGCCTT
TAA
Mutationat
position7
BG32
54TT
AAAGGCCGCTT
TTGCGGGATC
GTC
ACCCTC
AGCAGCGAGAGACAGCATC
GGAACGAGGGTA
GC
Mutationat
position7
BG32
55GCTA
CCCTC
GTT
CCGATG
CTG
TCTT
CCGCTG
CTG
AGGGTG
ACGATC
CCGCAAAAGCGGCCTT
TAA
Mutationat
position8
BG32
56TT
AAAGGCCGCTT
TTGCGGGATC
GTC
ACCCTC
AGCAGCGGAAGACAGCATC
GGAACGAGGGTA
GC
Mutationat
position8
BG32
66GCAGAACAATG
GTT
ACTT
TTTC
GATA
CGTG
AAACATG
TCCCACGGTA
GCCCAAAGACTT
GAGAGT
Ran
dom
target
BG32
67ACTC
TCAAGTC
TTTG
GGCTA
CCGTG
GGACATG
TTTC
ACGTA
TCGAAAAAGTA
ACCATT
GTT
CTG
CRan
dom
target
BG33
31GCTA
CCCTC
GTT
CCGATG
CTG
TCTT
TTGCTG
CTG
AGGGTG
ACGATC
CCGCAAAAGCGGCCTT
TAA
Mutationat
position9
BG33
32TT
AAAGGCCGCTT
TTGCGGGATC
GTC
ACCCTC
AGCAGCAAAAGACAGCATC
GGAACGAGGGTA
GC
Mutationat
position9
BG33
33GCTA
CCCTC
GTT
CCCATG
CTG
TCTT
TCGCTG
CTG
AGGGTG
ACGATC
CCGCAAAAGCGGCCTT
TAA
Mutationat
position-4
BG33
34TT
AAAGGCCGCTT
TTGCGGGATC
GTC
ACCCTC
AGCAGCGAAAGACAGCATG
GGAACGAGGGTA
GC
Mutationat
position−4
BG33
65GCTA
CCCTC
GTT
CCGATG
CTG
TCTT
TCTC
TGCTG
AGGGTG
ACGATC
CCGCAAAAGCGGCCTT
TAA
Mutationat
position10
BG33
66TT
AAAGGCCGCTT
TTGCGGGATC
GTC
ACCCTC
AGCAGAGAAAGACAGCATC
GGAACGAGGGTA
GC
Mutationat
position10
BG33
67GCTA
CCCTC
GTT
CCGATG
CTG
TCTT
TCGCTG
CAGAGGGTG
ACGATC
CCGCAAAAGCGGCCTT
TAA
Mutationat
position15
BG33
68TT
AAAGGCCGCTT
TTGCGGGATC
GTC
ACCCTC
TGCAGCGAAAGACAGCATC
GGAACGAGGGTA
GC
Mutationat
position15
BG33
69GCTA
CCCTC
GTT
CCGATG
CTG
TCTT
TCGCTG
CTG
AGGGTG
ATG
ATC
CCGCAAAAGCGGCCTT
TAA
Mutationat
position24
BG33
70TT
AAAGGCCGCTT
TTGCGGGATC
ATC
ACCCTC
AGCAGCGAAAGACAGCATC
GGAACGAGGGTA
GC
Mutationat
position24
BG33
71GCTA
CCCTC
GTT
CCGATG
CTG
TCTT
TCGCTG
CTG
AGGGTG
ACGATC
CAGCAAAAGCGGCCTT
TAA
Mutationat
position30
BG33
72TT
AAAGGCCGCTT
TTGCTG
GATC
GTC
ACCCTC
AGCAGCGAAAGACAGCATC
GGAACGAGGGTA
GC
Mutationat
position30
Plasmid
construction
BG32
18GGCCCGGATC
CGTC
GGGCGAGCGATG
ATG
CG
lambdafrag
men
t+Bam
HI(fw)
BG32
19CGCGCAAGCTT
CATC
GGCGTT
TCATT
CCCGTT
Tlambdafrag
men
t+HindIII
(rv)
Mutantplasm
idsequen
cing
BG24
55TT
TCCCAGTC
ACGACGTT
GJ3
protospacer
forw
ardprimer
BG24
56GGATA
ACAATT
TCACACAGG
J3protospacer
reve
rseprimer
Mutationsareindicated
inbold
italics.
Theprotospacer
regionis
underlin
ed.
PAM
isindicated
initalics.
*Only
oneoftw
oco
mplemen
tary
primersusedformutagen
esis
isshown.
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