Supplemental Material to - RUA

Landes Bioscience

www.landesbioscience.com

Supplemental Material to: César Díez-Villaseñor, Noemí M. Guzmán, Cristóbal Almendros, Jesús García-Martínez and Francisco J.M. Mojica CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli 2013; 10(5) http://dx.doi.org/10.4161/rna.24023 http://www.landesbioscience.com/journals/rna/article/24023

Figure S1. Genetic elements related to the CRISPR I-E loci of E. coli. Nucleotides at the PAM as well as the spacer-CRISPR and CRISPR-leader junctions are shown (W= A or T). Sequences used throughout the text to define each component correspond to the strands labeled with an asterisk.

Leader-A CCTGCGCGTCAAACCATTCTTTCGGTGATACACAT GAGTCGACGTTTATCAATATCAGTTGCACCGGCAT GGGATTATCAACGAGGTCAAATAATTTACTGCCAT ACATGAATGTCGGTTCAGACCGTGTTTTTACCCAT CGTCCGGATCGGTTTCGAGAATCTCTACGCTCCAT TTTTGCTGACACCGGCAATACTGAACGGCTGGCAC ATAATCGCCCAGGCGCGAAACGCCTCGAACGACTT ATCTCCAGCGCCTTTTCGACGCAACGGTGCGCCAT TGACCGATTTTATTGATCGCCCGATAGACCTGCAT CACATTCAACAGGTTAAGGTAACCGATTTGACCAT ATTTTCCTGATCGAGTTTTGAGAATTTTGTCGCAT CCCAGACCTGAAGTCAACTTATCCATTGTCGACTT AAACCGAAACACACGATCAATCCGAATATGAGCAT GTTTCCCGTGCGTCGTATGAGGCAGAAAAGAGCAT CGCGTAAGGTGTGCCAGCTATAAAAAACTCACCAT ATCGTTCTTGATGCCATGGTGGCGTGTAAGCCCAT CTGTTGGCAAGCCAGGATCTGAACAATACCGTCGT CAAAGGACACCGGGAGGCACCCGGCACCGCAGCCT TGCCGCGAAGACCAGTGAACTGGAAATCTGGCCAT GCGCGGCATGTGAATAGATGGACGCCAGGCACCAT GCTGGTGGCGCGGGCAAACGGAACAATCCCGCCAT TTCTGATACGTACTGAGGCCGGTTGTGATACGCAT TTCCTGTGCCCTGAGTTGCACCTGTTGTTCCGCTT TCGTCCCTGGGAGCGGCTTCAAACCACTCTCTCAT TGAAATGCTGCCGGAGGTAAAACCAGAAACAGCAT AAACATATGCACCGGGTCTCAGTGGGAATGATCAT TTTCTAGGAAACGACGTTATTTGCTCATCCCACAT TCGATCGCAAATATGCTCCGATTGATGAAAAGCTT TGGCTAAGAGCAAAAACAGGGGTATGCAGACGCAT CTTTGACCGAGTCTGATATTGCCCGTGCCTGCAAG CAAAAAATAAAATAAAGGCAATGTTTTCAATGCAT TCATACTGCTCCCACACCGAAAGCGCGGCAGCCAT TTCACCATCAACGCCCGTTTCGAAATTAATTTCCT ACGATGGCGATGCGTGAGAAAGGGGGTCGATACGT AGAAATCAAACTCAGCGTGCAAATGGGGTAGCCAT GCGGGCGGATTCAATCTGGCCAATATCGACGAGCT TTTACAGCTACGCCCATGTAGGCCACCTGCACCAT TTATTTGACCTCGTTGATAATCCCACAGATCCATC ACTGGCGTTGAGGCGTGGAAGCCTAATTTTTATAT TATCCAGTACCCGCCAGCGGGTTTACTGACGGCAT GGCTGCTGGCGATGGCATATATGGCAATTGGCTGG GCGAACGGATTCATGCGCCACTGCGCCGCCTGCAT ACCGCATTAATTCACGAGACGTATCGCGGAGATAT GAGCCCTGCCAGAATGGGGCCTCTTTTGTACCCAT CCTGATTGATGGCTTCTTTGATGTCAAACCGACAT ATCGACTTATGCCCCATCAGGCTCTGCAATACCTT AGCACGGCAGGCCATATGAAATACCTGTTGCTCTT GCACCCATCAAGGAATAACGCGTTTATTTTCCCAT GAAACGCCGGTTGAACGTCGTGCAAAAATCATCAT CACGACGGGCAGGCGATCACCGGCATGACCCACAT TTGCTGCAATTTTGTCTTTTAATTTATCCTAGCTT GTCGATAATATTTTGCTGTTGTTTTGTTACGTCTT ATTAAATCGTCAGAAAATAGCGGTAATCAAGTCAT TGCTCCGGAGATAACGAATATAAAAATGACGCCAG AGTTGTGATACGCATTGCACTCTCCTGAACCTCTT

Leader-T AAGACAACGCGTTCGGATGTTTTCTGGCTGGGCTT CAATACGACCGCGAATAAACATGATGTGATCAGCG CATTAATTGGGAACGCCTCATCACTGCCACCGCTT GCATCAGTACCCGCTCAAAGCTCTCCATCACCCTT TCTGACGCCGGATACTCCCCGGGAGCGATTTTCTT CTCACCATCAACACCCGTTTCAAAATTAATTTCCT ATTAACCCGCCGTGCGCGGGTTTTTTTGTGCCTGT GGCTGCGAAATACGGCTACGACATCTCTGGTCCGG ATCCCCTCCCAAAGTTTTTTCATGTTTTCTGGCTT GCCGTCCAGAATTTTTTAAAGCGCTTCAACTGCTT GTTATTCGTCGCGCGGCGGCTCATGTCGATCTCAT CAGAACGCGGCGCAGCTCGTCACGCAAAAATGCTC TATCCAGCGCTCAATCTGGAAATTAAACTCCTCCT TTTTTTTCCAGGAATTCATTTTCCTGATCGAGTTT AGAGCAACAGGTATTTCAAATGGCCAGCCGTGCTT TGTTGATGGTGAGTTTTTTATAGCTGGCGCACCTT TTTACGTCGTTGATGACATCGTTCAGGTGTTTCTT ACTGGCGCTGGCGGGGCGGCGTAGAGCGGTAAATA GGTTCAAATCCTCTCGTGCCGACCAAAAACACCAA ACATCCTGACCAGCTGTGGGTGGCTGATTTTACTT TCCGGTACAAAAGATGGCCTCTATATTTTTGACTT

Figure S2. Sequence of protospacer regions of E. coli CRISPR2 spacers used to generate the WebLogos shown on Figure 3A (Leader-T) and 3B (Leader-A). PAM positions are shadowed.

CRISPR2.1 Array

Strain IAI1

CRISPR2.3 Array

AC

EC7

AT

EC1

AT

EC6

AT

AC

AT AT

AT AT

TT

AT AT

AT GT CT

CR

L

A

A

CR

A

A

A

A

A

A

A

A

AT

A A

EC44

AT

A

A

E042

AT

CT

A

A

T

EC47

AT

AT

A

A

T

UMN026

AT

AT

A

A

T

A

A

T

A

A

A

A

A

A

A

A

T

A

A

T T

B171

AT

GT

E22

GT

EC49

AT

EC50

AT

TT

EC31

AT

EC43

AT

AT

AT

T

T TT

TT

T

TT AT

T

EC37

AT

A

A

O157:H7

AT

A

A

T

A

A

A

EC34

AT AT

EC42

AT

EC46

GT AT

AT

EC69 E24377A

A

A

A

A

A

A

A

A

EC35

TT

A

A

EC36

TT

A

A

A

A

T

T

T

T

T

T

T

T

SMS-3-5

AT

CT

EC4 EC15

AT

EC16

AT

TT

EC18

CG

8739

TT

T T

AT

A TC

AG

A TT

TT

GT CT GG

TT

T

AT TT TT

TC TT

TT GT CT TT TT TT

TT

EC19

T

T

EC20

T

T

EC22

T

T

TT

GT CT

T

T

TT

TT

A T

TT

T

TT TT

A

AT

T

53638

TT

A GG

T

TT

TT

AT

T

HS

CG

AT

TT

CT

CT

A

TA AA

T

TT

T

CT

A TT GG

A TT

Figure S3. Correlation between PAM, CRISPR and leader sequences along CRISPR arrays of representative E. coli strains. CRISPR2.1 and CRISPR2 3 arrays are shown equally oriented with respect to the leader (right). CRISPR2.3 (right) Spacers are represented by boxes (repeats are omitted), omitted) and when similar sequences were identified in non-CRISPR loci, the 2nd and 3rd nucleotides of the PAM region are indicated. Identical spacers are labeled with the same color (white boxes correspond to strain specific spacers). The variants (A or T) of the leader2.1 (L) and the predominant CRISPR (CR) of each array are indicated.

T A

Exponential

Stationary

2μg 5μg 10μg 2μg 5μg 10μg

C1

C2

pWUR399 (t) Chromosome pWUR399 (d) pCSIR-T (t) pCSIR-T (d) pWUR399 (m)

pCSIR-T (m)

Figure S4. Agarose gel electrophoresis of total DNA samples extracted from cultures of BL21-AI strain carrying plasmids pWUR399 and pCSIR-T, grown as in the acquisition assays. About 2, 5 and 10 µg of extractions from exponential or stationary phase cultures were run. The chromosomal and supercoiled plasmid bands corresponding to monomer (m), dimer (d) and trimer (t) molecules are indicated. Total DNA samples (10 µg) from BL21-AI carrying either pWUR399 (C1) or pCSIR-T (C2) are also included.

>pCSIR-T CACCTTTGGCTTCGGCTGCGGTTTATCCCCGCTGGCGCGGGGAACTCTGCGTGAGCGTATCGCCGCGCGTCTGCGAAAGCGGTTTATCCCC GCTGGCGCGGGGAACTCTCTAAAAGTATACATTTGTTCTTAAAGCATTTTTTCCCATACAAACAACCCACCAACCTTAATGTAACAAAAGC CGAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATCGCATCGTAAA GAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGA AAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGG TGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCAC GACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGA ATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTT CACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTC GGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATTTTTTTAAGGCAGTTCTAGA

>pCSIR-A AAGCTTACCGCAGCAAATTACGGTTCCAGAAACGCGCGGTTTATCCCCGCTGGCGCGGGGAACACTTCACGCCAACGCAGAGGCGGGCCGC GTTAATCGGTTTATCCCCGCTGGCGCGGGGAACACACTAAGCATACATATCTGTTTTTAAACAAATTTATTCCACATCTCGAGAAAAAAAT CACTGGATATACCACCGTTGATATATCCCAATCGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAG ACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCC TGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCA TGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGT TACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTG ATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCT GGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGC GGGGCGTAATTTTTTTAAGGCAGTTCTAGA

>pCas1-2[K] CCATGGCCTGGCTTCCCCTTAATCCCATTCCACTCAAAGATCGCGTCTCCATGATCTTTCTGCAATATGGGCAGATCGATGTAATAGATGG CGCGTTTGTACTTATCGACAAGACAGGGATCCGCACTCATATTCCTGTTGGCTCGGTTGCCTGCATCATGCTGGAACCTGGTACACGGGTT TCGCATGCAGCTGTACGCCTGGCTGCGCAAGTTGGAACATTGTTGGTATGGGTGGGGGAAGCGGGCGTTCGTGTTTATGCTTCTGGTCAGC CTGGAGGTGCGCGTTCAGATAAGCTGCTCTATCAGGCAAAACTTGCTCTGGATGAAGATTTGCGTCTGAAGGTCGTACGTAAAATGTTTGA ACTTCGGTTTGGAGAACCTGCGCCTGCCCGGCGCTCCGTAGAGCAACTCAGAGGTATAGAAGGCAGTCGCGTGCGGGCAACCTACGCACTT CTGGCGAAGCAATACGGCGTGACATGGAATGGACGTCGCTACGATCCGAAAGACTGGGAAAAGGGCGATACGATCAACCAATGCATTAGCG CTGCAACTTCCTGTTTATACGGCGTAACTGAAGCGGCGATACTTGCAGCTGGTTATGCACCAGCTATTGGGTTTGTGCATACAGGAAAGCC TCTTTCCTTTGTTTACGATATTGCAGACATCATTAAATTTGACACTGTTGTACCGAAAGCTTTTGAGATAGCGCGTCGTAACCCTGGTGAG CCGGACCGGGAAGTCCGTTTGGCGTGCAGGGATATTTTTCGCAGTAGTAAAACATTAGCCAAATTGATTCCGCTTATAGAGGACGTGCTTG CCGCTGGAGAAATACAACCGCCGGCCCCACCTGAAGATGCACAGCCTGTTGCCATTCCGCTTCCTGTTTCACTGGGAGATGCAGGCCATCG GAGTAGCTGAAATGAGTATGTTGGTCGTGGTCACTGAAAATGTACCTCCGCGCTTACGAGGCAGATTAGCCATCTGGTTGTTGGAGGTACG TGCAGGGGTATATGTAGGTGATGTATCCGCAAAAATTCGTGAAATGATCTGGGAACAAATAGCTGGACTGGCGGAAGAAGGCAATGTAGTG ATGGCATGGGCAACGAATACGGAAACGGGATTTGAGTTCCAGACATTTGGGTTAAACAGGCGTACCCCGGTAGATTTGGATGGTTTAAGGT TGGTGTCTTTTTTACCTGTTTGAGCGGCCGC

>pCas1-2[O] CCATGGCGTTTGTACCACTGAGTCCGATCCCGTTAAAAGATCGCACCTCTATGATCTTCCTCCAGTACGGTCAAATCGACGTACTGGACGG CGCTTTCGTGCTGATCGACAAAACCGGGATCCGCACGCACATTCCGGTGGGATCGGTCGCCTGCATTATGCTCGAACCGGGAACGAGAGTT TCCCACGCGGCGGTGCATCTGGCCGCCACGGTGGGAACACTGCTGGTCTGGGTCGGTGAAGCGGGCGTTCGCGTTTACTCTTCCGGACAAC CCGGAGGGGCGCGGGCAGATAAATTACTCTACCAGGCAAAGCTGGCTTTAACGGAAGATCTACGCCTGAAGGTGGTGCGCAAAATGTATGA ATTACGTTTTCGTGAGCCACCGCCAGCTCGCTGTTCAGTGGATCAGCTACGGGGAATTGAGGGATCCCGCGTTCGCCAGACCTATGCATTA CTGGCGAAACAATATGGTGTGAAATGGAATGGTCGCAAATACGATCCTAAAGACTGGGAAAAAGGCGATGTTGTGAATCGCTGCATCAGTG CTGCCACATCATGTCTGTACGGTATTTCTGAAGCGGCAGTATTAGCCGCGGGATATGCGCCCGCTATTGGATTTATTCATAGTGGCAAACC GCTTTCATTTGTTTATGACATAGCCGATATCATTAAATTTGATTCGGTTGTGCCAAAGGCATTTGAAATAGCAGCGAGGCAACCCGCAGAA CCTGATAAAGAAGTCAGATTAGCCTGTCGCGATATTTTCCGTAGCACTAAGTTAACGGGCAAATTAATACCGTTAATTGAGGAAGTCCTTG CTGCAGGTGAAATTGAACCACCACAACCCGCGCCGGATATGTTACCGCCTGCCATCCCTGAACCTGAAACGCTGGGTGATAGTGGTCACCG GGGGCGCGGCGGATGAGCATGGTCGTGGTTGTTACAGAAAATGTCCCTCCGCGCTTACGTGGACGGCTCGCAATCTGGCTACTGGAAGTGC GTGCCGGTGTGTATGTTGGTGATACATCAAAACGTATTCGGGAGATGATCTGGCAGCAAATTACCCAACTGGCTGGTTGCGGAAATGTGGT GATGGCCTGGGCGACCAATACCGAGTCGGGTTTTGAATTTCAGACCTGGGGAGAAAACAGACGTATTCCGGTGGATTTGGATGGGTTACGT TTGGTTTCTTTTCTTCCTGTTGATAATCAATAGGTTATGTGTTCTTTAAAAATAAGGAAATGTTTGAATTTAGTTGGTAGATTGTTGATGC GGCCGC

Figure S5. Sequences of fragments cloned in plasmids pCSIR-T, pCSIR-A, pCas1-2[K] and pCas12[O]. Restriction sites used for cloning are underlined and CRISPR duplicons (in pCSIR-T and pCSIR-A) as well as cat (in pCSIR-T and pCSIR-A) and cas1-cas2 genes (in pCas1-2[K] and pCas12[O]) shadowed.

Table S1. Spacers integrated into pCSIR-T during propagation in BL21-AI harboring pWUR399.

a

Spacer Id. 1 2 3 4 5 6 7 8

Spacer sequence (5’-3’) GAGATCCCGGACACCATCGAATGGCGCAAAACC ATTCGATGGTGTCCGGGATCTCGACGCTCTCCC ATCGCCGCTTCAGTTACGCCGTATAAACAGGAA GTCGAGATCCCGGTGCCTAATGAGTGAGCTAAC TCTTGCAGGTATCTTCGAGCCAGCCACGATCGA TAACACCCCATGCAACGATGGTTATCTCAGAAC CCCTGCCCTGAACCGACGACCGGGTCATCGTGG GGCGCGGGGCATGACTAACATGAGAATTACAAC

Times observed 2 1 1 1 1 1 1 1

Protospacer carrier

SAM

pWUR399 pWUR399 pWUR399 pWUR399 pWUR399 pWUR399 pWUR399 pWUR399

CTT CTT AGT CTT ACAa CTT GCC CTT

Thymine is found at position 4 after the protospacer, conforming a putative one-position shifted CAT SAM.

Table S2. Spacers integrated into pCSIR-T during propagation in K12 harboring pCas1-2[K].

a

Spacer Id. 1 2 4 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53

Spacer sequence (5’-3’) GAGATCCCGGACACCATCGAATGGCGCAAAACC ATTCGATGGTGTCCGGGATCTCGACGCTCTCCC GTCGAGATCCCGGTGCCTAATGAGTGAGCTAAC GCGCAGCAGTCGGGCTGAACGGGGAGTTCTTGC TATTGGGCGCCAGGGTGGTTTTTCTTTTCACCA CCCATATTGCAGAAAGATCATGGAGACGCGATC TCGACAATGTTCAACAGCCACGGAATATCTTTC AAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGC GACCAGATGCTCCACGCCCAGTCGCGTACCGTC TGAACAGGATCTATTTGAGGCGCTAAATGAAAC TGCATTGGTTGATCGTATCGCCCTTTTCCCAGT ATGTCTGCAATATCGTAAACAAAGGAAAGAGGC TTGCGCTCTCCTGTTCCGACCCTGCCGTTTACC CGTATTCGTTGCCCATGCCATCACTACATTGCC ATCCGCTCATGAGACAATAACCCTGATAAATGC GTCCGCACCAACGCGCAGCCCGGACTCGGTAAT CCGAGTACGTGCTCGCTCGATGCGATGTTTCGC ACATTGCCTTCTTCCGCCAGTCCAGCTATTTGT ATGGGTGGGGGAAGCGGGCGTTCGTGTTTATGC CGCCATTACCGAGTCCGGGCTGCGCGTTGGTGC AAGAAATAACGCCGGAACATTAGTGCAGGCAGC GATACTTCGGCGATCACCGCTTCCCTCATACTC CAATACGCAAACCGCCTCTCCCCGCGCGTTGGC TGAGAATTACAACTTATATCGTATGGGGCTGAC CCGTTTACCGGATACCTGTTCCGCCTTTCTCCC TGTACCGAAAGCTTTTGAGATAGCGCGTCGTAA TAGAAGGCAGTCGCGTGCGGGCAACCTACGCAC CGATACGATCAACCAATGCATTAGCGCTGCAAC GCCCCTCGGCTTGAACGAATTGTTAGACATTAT GAACGAATTGTTAGACATTATTTGCCGACTACC GTATCAGCCCGTCATACTTGAAGCTAGACAGGC CGGAATGATGTCGTCGTGCACAACAATGGTGAC CCGGGATCTCGACGCTCTCCCTTATGCGACTCC CTCACCAGGGTTACGACGCGCTATCTCAAAAGC GATTAAGGGGAAGCCAGGTCATGGTATATCTCC CGCTTCCCCCACCCATACCAACAATGTTCCAAC CCGCTGGACCTACCAAGGCAACGCTATGTTCTC TTACGCGCAGAAAAAAAGGATCTCAAGAAGATC CTTCGGCGATCACCGCTTCCCTCATACTCTTCC GTTATACGATGTCGCAGAGTATGCCGGTGTCTC ATTGGGTTTGTGCATACAGGAAAGCCTCTTTCC GTCAAAAGCCCCACTTATACGCTGGTCGAACCC GTATGGGTGGGGGAAGCGGGCGTTCGTGTTTAT TTTGCATGGTTTGTTGAAAACCGGACATGGCAC TCGACCAAGAAGCGGTTGTTGGCGCTCTCGCGG ATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGC AGCCAGCCAGACGCAGACGCGCCGAGACAGAAC CCCAGTGAAACAGGAAGCGGAATGGCAACAGGC

Times observed 2 2 2 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 3 1 2 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 1 1 2 1 1 1 1 1 1 1 1 1

Protospacer carrier

SAM

pCas pCas pCas pCas pCas/Chromosome pCas/Chromosome Chromosome pCSIR pCas/Chromosome pCas pCas/Chromosome pCas/Chromosome pCas pCas/Chromosome pCas pCas pCSIR pCas/Chromosome pCas/Chromosome pCas/Chromosome pCas/Chromosome pCas pCas/pCSIR/Chromosome pCas pCas pCas/Chromosome pCas/Chromosome pCas/Chromosome pCas pCas pCas pCas pCas pCas/Chromosome pCas pCas/Chromosome pCas pCas pCas pCas pCas Chromosome pCas/Chromosome pCas/Chromosome pCSIR pCas/Chromosome pCas/Chromosome pCas/Chromosome

CTT CTT CTT CTT AGT CTT CTT CTA CTT CCTa TCTa CTT CGG CTT CTT TGG CTT TTC CTT CTT CTT CTT CCG CTT CTT ACC CTT CTT TTT CTT CTT CTT CTG CTT CTT CTT CTT CTT CTT CTT CTT CTA TGC CTC GCTa CTG CTT CTG

Thymine is found at position 4 after the protospacer, conforming a putative one-position shifted CTT SAM.

Table S3. Spacers integrated into pCSIR-A during propagation in O157:H7 harboring pCas1-2[O].

a

Spacer Id. 4 8 9 14 22 26 27 29 35 37 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Spacer sequence (5’-3’) GTCGAGATCCCGGTGCCTAATGAGTGAGCTAAC GGCGCGGGGCATGACTAACATGAGAATTACAAC GCGCAGCAGTCGGGCTGAACGGGGAGTTCTTGC GACCAGATGCTCCACGCCCAGTCGCGTACCGTC CCGAGTACGTGCTCGCTCGATGCGATGTTTCGC AAGAAATAACGCCGGAACATTAGTGCAGGCAGC GATACTTCGGCGATCACCGCTTCCCTCATACTC TGAGAATTACAACTTATATCGTATGGGGCTGAC GAACGAATTGTTAGACATTATTTGCCGACTACC CGGAATGATGTCGTCGTGCACAACAATGGTGAC CATTCGATGGTGTCCGGGATCTCGACGCTCTCC ACGACCATGCTCATCCGCCGCGCCCCCGGTGAC GCAATGCGCGCCATTACCGAGTCCGGGCTGCGC CCCACAGATTCCGTGGATAAGCAGGGGACTAAC ACTTACAAGTGGATCGGCATTGGTGATTACACC TTCACCAGTGAGACGGGCAACAGCTGATTGCCC CCACAACCCGCGCCGGATATGTTACCGCCTGCC ATTACCGAGTCCGGGCTGCGCGTTGGTGCGGAC TCCTCAATTAACGGTATTAATTTGCCCGTTAAC TTCAGTGACAACGTCGAGCACAGCTGCGCAAGG TGAACCACCACAACCCGCGCCGGATATGTTACC GGACTCAGTGGTACAAACGCCATGGTATATCTC TTCCATAGGCTCCGCCCTCCTGCCAGAGTTCAC AATTGAGGGATCCCGCGTTCGCCAGACCTATGC GCAGTGGGAACGATGCCCTCATTCAGCATTTGC GAATGGCAGCGCAATGACATTCTTGCAGGTATC CAGATGCTCCACGCCCAGTCGCGTACCGTCTTC GTCCCCTGCTTATCCACGGAATCTGTGGGTAAC CTCATCCGCCGCGCCCCCGGTGACCACTATCAC AGCATCGCAGTGGGAACGATGCCCTCATTCAGC ATTGTGCACCGCCGCTTTACAGGCTTCGACGCC GATCCTTGGCGGCAAGAAAGCCATCCAGTTTAC

Times observed 6 1 1 2 1 1 1 1 1 2 1 1 1 2 1 2 1 1 3 1 1 1 2 1 1 1 1 2 1 1 1 1

Protospacer carrier

SAM

pCas pCas pCas pCas/Chromosome pCSIR pCas pCas pCas pCas pCas pCas pCas pCas/Chromosome pCas Chromosome pCas/Chromosome pCas/Chromosome pCas pCas pCSIR pCas/Chromosome pCas pCas pCas/Chromosome pCas pCas pCas pCas Chromosome pCas pCas pCSIR

CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CCTa CCA CGT CAT CAT CTT CAT CAT CTT GAA CGC CCTa CAT CAT CAT CTT CAT CTT CCC CAT CGC CTT

Thymine is found at position 4 after the protospacer, conforming a putative one-position shifted CTT SAM.

Table S4. Plasmids constructed in this study. Name pCR2.1-Cm pCSIR-A

Size (bp) 4,592 4,662

CTT/ Kb 35.9 35.8

CAT/ Kb 27.9 27.9

Vector

pCSIR-T

4,770

36.1

27.7

pCR2.1

pCas1-2[O] pCas1-2[K]

4,777 4,711

26.8 29.9

28.7 30.1

pCDF-1b pCDF-1b

pCR2.1 pCR2.1-Cm

Cloning site(s) XhoI+XbaI HindIII+XhoI EcorI XhoI+XbaI NcoI+NotI NcoI+NotI

Insert(s)

Primers

Template

cat ORF type A CRISPR cassette type T CRISPR cassette cat ORF cas1- cas2 variant E-O cas1- cas2 variant E-K

CATF1/CATR1 AS2F /ALR

pKK232-8 ECOR69

TS2F/ TLR

K12

CAS1EOF /CAS2EOR CAS1EKF/ BG237530

O157:H7 pWUR399

Table S5. Spacers integrated into pCSIR-A during propagation in K12 harboring pCas1-2[K].

a

Spacer Id.a 1 1' 4R 20R 24 24' 24R 29 37R 76 77 78 79 80 81 82 83

Spacer sequence (5’-3’) GAGATCCCGGACACCATCGAATGGCGCAAAACC aCGAGATCCCGGACACCATCGAATGGCGCAAAACCb GTTAGCTCACTCATTAGGCACCGGGATCTCGAC GCATTTATCAGGGTTATTGTCTCATGAGCGGAT ATGGGTGGGGGAAGCGGGCGTTCGTGTTTATGC acATGGGTGGGGGAAGCGGGCGTTCGTGTTTATGCb GCATAAACACGAACGCCCGCTTCCCCCACCCAT TGAGAATTACAACTTATATCGTATGGGGCTGAC GTCACCATTGTTGTGCACGACGACATCATTCCG TCATCCAGAGCAAGTTTTGCCTGATAGAGCAGC GGCAGATCGATGTAATAGATGGCGCGTTTGTAC ACCGACGACCGGGTCATCGTGGCCGGATCTTGC GCGGTATGGCATGATAGCGCCCGGAAGAGAGTC GAATGTAATTCAGCTCCGCCATCGCCGCTTCCA GTTTTCCGTAGCGTGCTTCCTGCCAGGCTGCGC acGCAGCGCTAATGCATTGGTTGATCGTATCGCCCb TTTCGCCTGCTGGGGCAAACCAGCGTGGACCGC

Times observed 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1

Protospacer carrier

SAM

pCas pCas pCas pCas pCas/Chromosome pCas/Chromosome pCas/Chromosome pCas pCas pCas/Chromosome pCas/Chromosome pCas pCas pCas Chromosome pCas/Chromosome pCas

CTT CTT CCGc TACc CTT CTT TACc CTT GTGc CTT CTT CGG CAA ACTd CTT CTT CTT

Spacers identified with #’ differ of the corresponding spacer # (integrated in this or another acquisition experiment) by the presence of AC di-nucleotide at the 5’ end. Spacers identified with #R are identical to the respective spacer # integrated in the reversed orientation in this or another acquisition experiment. b 35 nt spacer. Mismatches with the most similar sequence (putative protospacer) are in lowercase letters. c 5’-CTT-3’ tri-nucleotide adjoins the opposite end of the protospacer in the complementary strand. d Thymine is found at position 4 after the protospacer, conforming a putative one-position shifted CTT SAM.

Table S6. Number of times CTT, CAT and non-CWT (Other) motifs were sampled in acquisition assays carried out in strains harboring plasmids with diverse cas1-2 and CRISPR/leader2.1 variant combinations. SAM cas1-2 variant CRISPR/Leader variant CTT CAT Other K T 49 0 20 K A 15 0 3 O A 26 11 8

Table S7. Primers used in this work.

a

Sequence (5’- 3’)c CTAAGGAAGCTAAAcTcGAG TTAAGGGCATcTaGaACTGC gcgcgccATGgCCTGGCTTCCCCTTAATCC ggcccgcggccgcTCAAACAGGTAAAAAAGACACCAAC cgcccaTGgCGTTTGTACCACTGAGTCC ccgcggCCgCATCAACAATCTACCAAC CACCTTTGGCTTCGGCTGC tTGTTACATTAAGGTTGGTGGGTTgTTTGTATGG GGAAagCtTACCGCAGCAAATTAC GGTAGAcTcgaGATGTGGAATAAATTTG CTAGTTATTGCTCAGCGG TAATACGACTCACTATAGGG CAGGAAACAGCTATGAC

Features XhoI site includes mutated cat start codon (CTC) cat stop codon (TAA) follows XbaI site NcoI site includes start codon (ATG) cas2 stop codon (TGA) follows NotI site NcoI site includes start codon (ATG) cas2 stop codon (TAG) follows NotI site

Primer used for cloning. Primer used for sequencing. Restriction sites are underlined and mismatches shown in lowercase.

b c

Name CATF1a CATR1 a CAS1EKF a BG2375 a CAS1EOF a CAS2EOR a TS2F a TLR a AS2F a ALR a T7tb T7b M13Rb

Includes mutations to avoid stop codons HindIII site XhoI site