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.

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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)

Semenova et al. www.pnas.org/cgi/doi/10.1073/pnas.1104144108 9 of 10

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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