Supporting Material

Supporting Material

Figure S1 – Individual plasmid loss assays. Individual plasmid loss graphs of C/G mutant pairs. Graphs show standard error of the mean (SEM) of two independent experiments with two replicates each.

1

Figure S2 – Individual Cas3 activity assays. Individual graphs of C/G mutant pairs showing plasmid degradation by Cas3. The standard error of the mean of two independent experiments is indicated.

2

Table S1: Detailed list of tested mutants crRNA sequence

C

T

G

A

C

G

A

C

C

G

G

G

T

C

T

C

C

G

C

A

A

G

T

G

G

C

A

C

T

T

T

T

Duplex position

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

DNA Target strand

G

A

C

T

G

C

T

G

G

C

C

C

A

G

A

G

G

C

G

T

T

C

A

C

C

G

T

G

A

A

A

A

Mutant/(predicted) behavior 1C / priming 2C / priming

Mutant sequences

C C

C

C

C C

3C / priming

C

4C / priming

C

1G / pred. stable 2G / pred. stable

G G

G

C G

G G

G

5G / stable

G G

8G / stable

A G G

9G / stable

G G G

5C / pred. priming 6C / pred. priming 7C / pred. priming 8C / pred. priming 9C / pred. priming

C C

A C C

C G G

4G / pred. stable

7G / stable

C

C

3G / pred. stable

6G / stable

C

C C C

*grey columns represent pinch point positions that do not base pair and are ignored

G G G

G

G G

G

G

G

G G

G G

G G

C C

C

C

G C

G C C

C C

C C

C

C

Table S2: Non-linear regression parameters of EMSA

Oligo duplex

Fit Quality R2

P-value

Parameters Amplitude

CT/WTNT GT/WTNT

0.97 0.94

0.00079 0.0036

0.72 +/- 0.048 0.12 +/- 0.033

WTT/WTNT WTT/CNT WTT/GNT

0.95 0.92 0.93

0.002 0.006 0.0049

1.18 +/- 0.17 1.14 +/- 0.16 0.95 +/- 0.16

Kd 13.73 +/- 3.51 101.98 +/62.07 46.3 +/- 18.15 23.3 +/- 10.56 38.8 +/- 18.43

Affinity ratio (Amp/ Kd) 0.053 0.0011 0.026 0.049 0.024

Table S3: Oligo nucleotides used in this study Name BG5301 BG5302 BG6577

Sequence AAGGTTGGTGGGTTGTTTTTATGG GGATCGTCACCCTCAGCAGCG

BG7157 BG7395

TTTGAGCTCTTATTTGCCGACTACCTTGGTGATCTC TTTTCTAGAAAAAGTGCCACTTGCGGAGACCCGGTCGTCA GCTTACATTCAAATATGTATCCGCTC TTTTCTAGAAAAAGTGCCACTTGCGGAGACCCGGTCGTCA GCGTACATTCAAATATGTATCCGCTC

BG7396 BG7397

BG8393 BG8394 BG8395 BG8396 BG8397 BG8398 BG8399 BG8400 BG8401 BG8402

ATGTCATTGCGCTGCCATTC

TTTTCTAGACAACCGGGGCAGATCATTAGTTGCACCGCGA TTCGACATTCAAATATGTATCCGCTC

TTTTCTAGAaagctgacgGccgggtcGccgcaagGggcactt GtACATTCAAATATGTATCCGCTC TTTTCTAGAaagcGgacgGccgggtctccgcaagtggcacGA ttACATTCAAATATGTATCCGCTC TTTTCTAGAaagctgacgaccgggtctccgcTGgGggcacttt GACATTCAAATATGTATCCGCTC TTTTCTAGAaagctgacgaccgggGcGccgcGagtggcacttt tACATTCAAATATGTATCCGCTC TTTTCTAGAaagctgacgCccgggtcCccgcaagCggcacttC tACATTCAAATATGTATCCGCTC TTTTCTAGAaagcCgacgCccgggtctccgcaagtggcacCAt tACATTCAAATATGTATCCGCTC TTTTCTAGAaagctgacgaccgggtctccgcTCgCggcactttC ACATTCAAATATGTATCCGCTC TTTTCTAGAaagctgacgaccgggCcCccgcCagtggcactttt ACATTCAAATATGTATCCGCTC TTTTCTAGAaagctgacgaccgggtcCccCcCCgtggcacttt CACATTCAAATATGTATCCGCTC TTTTCTAGAaagctgacgCccgggCcCccgcaagtCgcacCtt tACATTCAAATATGTATCCGCTC

Description Primer fw for CRISPR array PCR Primer rv for CRISPR array PCR Sequencing primer for protospacers (StrepR) StrepR fw SacI PCR primer StrepR+sp8 protospacer PCR primer StrepR+mutPAMsp8 protospacer PCR primer StrepR+scrambled protospacer StrepR rv XbaI + mut1C sp8 protospacer StrepR rv XbaI + mut2C sp8 protospacer StrepR rv XbaI + mut3C sp8 protospacer StrepR rv XbaI + mut4C sp8 protospacer StrepR rv XbaI protospacer

+

mut1G

sp8

StrepR rv XbaI protospacer

+

mut2G

sp8

StrepR rv XbaI protospacer

+

mut3G

sp8

StrepR rv XbaI protospacer

+

mut4G

sp8

StrepR rv XbaI protospacer

+

mut5G

sp8

StrepR rv XbaI protospacer

+

mut6G

sp8

BG8403 BG8404 BG8405 BG8406 BG8407 BG8408 BG8409 BG8410

BG9069

BG9070

BG9071

BG9072

TTTTCTAGAaagctgCcgaccggACctccgcaagtggcCcttC tACATTCAAATATGTATCCGCTC TTTTCTAGAaagctgacgCccgggCctccgcaagtggcacCttt ACATTCAAATATGTATCCGCTC TTTTCTAGAaagctgacgCccgggtctccgcaCgtggcCctttt ACATTCAAATATGTATCCGCTC TTTTCTAGAaagctgacgaccgggtcGccGcGGgtggcacttt GACATTCAAATATGTATCCGCTC TTTTCTAGAaagctgacgGccgggGcGccgcaagtGgcacGt ttACATTCAAATATGTATCCGCTC TTTTCTAGAaagctgGcgaccggAGctccgcaagtggcGctt GtACATTCAAATATGTATCCGCTC TTTTCTAGAaagctgacgGccgggGctccgcaagtggcacGtt tACATTCAAATATGTATCCGCTC TTTTCTAGAaagctgacgGccgggtctccgcaGgtggcGctttt ACATTCAAATATGTATCCGCTC ACTCCAAGCGACCGGAGGCTTTTGACTTGATCGGCACGTA AGAGTCTAGAaagctgCcgaccggACctccgcaagtggcC cttCtACATTCAAATATGTATCCGCTCATGAGACAATAAC CCTGATAAATGCTTC GAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACAT ATTTGAATGTaGaagGgccacttgcggagGTccggtcgGc agcttTCTAGACTCTTACGTGCCGATCAAGTCAAAAGCCT CCGGTCGCTTGGAGT ACTCCAAGCGACCGGAGGCTTTTGACTTGATCGGCACGTA AGAGTCTAGAaagctgGcgaccggAGctccgcaagtggcG cttGtACATTCAAATATGTATCCGCTCATGAGACAATAAC CCTGATAAATGCTTC GAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACAT ATTTGAATGTaCaagCgccacttgcggagCTccggtcgCc agcttTCTAGACTCTTACGTGCCGATCAAGTCAAAAGCCT CCGGTCGCTTGGAGT

StrepR rv XbaI protospacer

+

mut7G

sp8

StrepR rv XbaI protospacer

+

mut8G

sp8

StrepR rv XbaI protospacer

+

mut9G

sp8

StrepR rv XbaI + mut5C sp8 protospacer StrepR rv XbaI + mut6C sp8 protospacer StrepR rv XbaI + mut7C sp8 protospacer StrepR rv XbaI + mut8C sp8 protospacer StrepR rv XbaI + mut9C sp8 protospacer

Mut7G sp8 non-target strand

Mut7G sp8 target strand

Mut7C sp8 non-target strand

Mut7C sp8 target strand

ACTCCAAGCGACCGGAGGCTTTTGACTTGATCGGCAC

BG9073

GTAAGAGTCTAGAaagctgacgaccgggtctccgcaagtggc acttttACATTCAAATATGTATCCGCTCATGAGACAAT AACCCTGATAAATGCTTC

BG9074

GAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACAT ATTTGAATGTaaaagtgccacttgcggagacccggtcgtc agcttTCTAGACTCTTACGTGCCGATCAAGTCAAAAGCCT CCGGTCGCTTGGAGT

BG9141 BG9142

GAAGCATTTATCAGGGTTATTG AAAGCCTCCGACCGGAGG

Sp8-WT non-target strand

Sp8-WT target strand pBex_50nt_flankprotospacer_fw pBex_50nt_flank-protospacer_rv

Table S4: Plasmids used in this study Plasmid pWUR925

Description Rhamnose inducible promotor – RFP, ColE1, KanR

source This study

pWUR926

pWUR925 backbone, sp8 protospacer, StrepR

This study

pWUR927

pWUR925 backbone, mutPAM sp8 protospacer, StrepR

This study

pWUR928

pWUR925 backbone, scrambled protospacer, StrepR

This study

pWUR929

pWUR925 backbone, mut1C sp8 protospacer, StrepR

This study

pWUR930

pWUR925 backbone, mut2C sp8 protospacer, StrepR

This study

pWUR931

pWUR925 backbone, mut3C sp8 protospacer, StrepR

This study

pWUR932

pWUR925 backbone, mut4C sp8 protospacer, StrepR

This study

pWUR933

pWUR925 backbone, mut1G sp8 protospacer, StrepR

This study

pWUR934

pWUR925 backbone, mut2G sp8 protospacer, StrepR

This study

pWUR935

pWUR925 backbone, mut3G sp8 protospacer, StrepR

This study

pWUR936

pWUR925 backbone, mut4G sp8 protospacer, StrepR

This study

pWUR937

pWUR925 backbone, mut5G sp8 protospacer, StrepR

This study

pWUR938

pWUR925 backbone, mut6G sp8 protospacer, StrepR

This study

pWUR939

pWUR925 backbone, mut7G sp8 protospacer, StrepR

This study

pWUR940

pWUR925 backbone, mut8G sp8 protospacer, StrepR

This study

pWUR941

pWUR925 backbone, mut9G sp8 protospacer, StrepR

This study

pWUR942

pWUR925 backbone, mut5C sp8 protospacer, StrepR

This study

pWUR943

pWUR925 backbone, mut6C sp8 protospacer, StrepR

This study

pWUR944

pWUR925 backbone, mut7C sp8 protospacer, StrepR

This study

pWUR945

pWUR925 backbone, mut8C sp8 protospacer, StrepR

This study

pWUR946 pWUR748

pWUR925 backbone, mut9C sp8 protospacer, StrepR

This study

pMat11-MBP-Cas3

(1)

pWUR868

pACYC poly spacer8 CRISPR array

(2)

pWUR514

cse2 with Strep-tag II (N-term)-cas7-cas5-cas6e in pET52b cse1 in pRSF-1b, no tags

(3)

pWUR408

(4)

References 1. 2. 3.

Mulepati, S. and Bailey, S. (2013) In Vitro Reconstitution of an Escherichia coli RNA-guided Immune System Reveals Unidirectional, ATP-dependent Degradation of DNA Target. The Journal of biological chemistry, 288, 22184-22192. Künne, T., Kieper, S.N., Bannenberg, J.W., Vogel, A.I., Miellet, W.R., Klein, M., Depken, M., SuarezDiez, M. and Brouns, S.J. (2016) Cas3-Derived Target DNA Degradation Fragments Fuel Primed CRISPR Adaptation. Mol Cell, 63, 852-864. Jore, M.M., Lundgren, M., van Duijn, E., Bultema, J.B., Westra, E.R., Waghmare, S.P., Wiedenheft, B., Pul, Ü., Wurm, R. and Wagner, R. (2011) Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nature structural & molecular biology, 18, 529-536.

4.

Brouns, S.J., Jore, M.M., Lundgren, M., Westra, E.R., Slijkhuis, R.J., Snijders, A.P., Dickman, M.J., Makarova, K.S., Koonin, E.V. and van der Oost, J. (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science, 321, 960-964.