Next Generation Bacterial Engineering: the CRISPR-Cas Toolkit

SUPPLEMENTALINFORMATION

Next generation bacterial engineering: the CRISPR-Cas toolkit

Ioannis Mougiakos1,#, Elleke F. Bosma1,#, Willem M. de Vos1, Richard van Kranenburg1,2, John van der Oost1

1Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands.

2Corbion, Arkelsedijk 46, 4206 AC Gorinchem, The Netherlands.

#Contributed equally.

*Correspondence: (J. van der Oost)

Table S1. Overview of CRISPR-Cas-based prokaryoticgenome editing.

Species / Cas type / HR methoda / Nr of plasmids used / sgRNA or crRNA:tracrRNA used / Multiplex yes/no / Ref
Escherichia coli / SpyCas9 / SSDR / 2 / crRNA:tracrRNA / no / [S1]
SpyCas9 / SSDR and DSDR / 2 / sgRNA / yes / [S2]
SpyCas9 / SSDR and DSDR / 3 / crRNA:tracrRNA / yes / [S3]
SpyCas9 / λRed recombineering-assisted Plasmid-HR / 2 / sgRNA / yes / [S4]
SpyCas9 / DSDR / 2 / sgRNA / no / [S5]
SpyCas9 / SSDR and DSDR / 2 / sgRNA / yes / [S6]
SpyCas9 / DSDR / 1 / sgRNA / yes / [S7]
SpyCas9 / None (aimed at cell killing) / 1 / crRNA:tracrRNA / yes / [S8]
SpyCas9 nickase / Chromosomal IS5 direct repeats and 2-4 target CRISPR system / 2 / sgRNA / yes / [S9]
Tatumellacitrea / SpyCas9 / λRed recombineering-assisted Plasmid-HR / 2 / sgRNA / yes / [S4]
Streptococcus pneumoniae / SpyCas9 / dsDNA based HR / 2 / crRNA:tracrRNA / no / [S1]
Clostridium beijerinckii / SpyCas9 / Plasmid-HR / 1 / sgRNA / no / [S10]
Streptomyces coelicolor / SpyCas9 / Plasmid-HR / 1 / sgRNA / no / [S11]
Streptomycescoelicolor / SpyCas9 / NHEJ and
Plasmid-HR / 1 / sgRNA / no / [S12]
Streptomyces
coelicolor / SpyCas9 / Plasmid-HR, combined with codA-counter-selection / 1 / sgRNA / no / [S13]
Streptomyces lividans / SpyCas9 / Plasmid-HR / 1 / sgRNA / yes / [S14]
Streptomyces viridochromogenes / SpyCas9 / Plasmid-HR / 1 / sgRNA / yes / [S14]
Streptomyces albus / SpyCas9 / Plasmid-HR / 1 / sgRNA / yes / [S14]
Lactobacillus reuteri / SpyCas9 / SSDR / 3 / crRNA:tracrRNA / no / [S15]
Clostridium cellulolyticum / SpyCas9-nickase / Plasmid-HR / 1 / sgRNA / no / [S16]
Sulfolobus islandicus / Native
Type I-A / Plasmid-HR / 1 / NA / no / [S17]
Sulfolobus islandicus Δcas3 / Native Type III-B Cmr-α / Plasmid-HR / 1 / NA / no / [S17]
Pectobacterium atrosepticum / Native
Type I-F / Genomic island reshuffling (AEJ) / 1 / NA / yesb / [S18]
Escherichia coli / Native
Type I-E / None (aimed at cell killing) / 3 / NA / no / [S19]

aAbbreviations: HR: homologous recombination; SSDR: single-stranded DNA recombineering (SSDR); DSDR: double-stranded DNA recombineering; plasmid-HR: plasmid-borne homologous recombination; NHEJ: non-homologous end-joining; AEJ: alternative end-joining

b Multiplexing was possible but not very effective, probably due to rearrangements of the repeats in the plasmid.

Table S2. Overview of CRISPR-Cas-based prokaryoticgene repression.

Species / CRISPR- Cas type / Max. fold
expression reduction / Nr of
plasmidsuseda / Required/modified genomic componentsa / sgRNA or crRNA:tracrRNAused / Multiplex yes/no / Ref
Escherichia coli / Spy-dCas9 / 100b / 1 / NA / tracrRNA:crRNA / no / [S20]
Spy-dCas9 / 300b
(multiplex 1000) / 2 / NA / sgRNA / yes / [S21]
Spy-dCas9 / 330b / 1 / conjugation machinery / sgRNA / no / [S22]
Spy-dCas9 / 100
(multiplex on different genes) / 1 / NA / sgRNA / yes / [S23]
Escherichia coli Δcas3 / Native Type I-E / Plasmid target: 900b
Genome target: 2200
(multiplex on different genes) / 1 / promoter replacement to activate Cascade / NA / yes / [S24]
Native Type I-E / Strain K12: 50b
Strain BL21: 15b / 2 / NA / NA / yes / [S25]
Streptococcus pneumoniae / Spy-dCas9 / 14b / 1 / dCas9 and tracrRNA integrated in genome / tracrRNA:crRNA / no / [S20]
Mycobacteria / Spy-dCas9 / Max. 94%
Multiplex test: 4-fold each vs. 16-fold multiplex / 1 / dCas9 integrated in genome / sgRNA / yes / [S26]
Bacteroides thetaiotaomicron / Spy-dCas9 / 45 / 1 / NA / sgRNA / no / [S27]
Streptomyces coelicolor / Spy-dCas9 / 100b / 1 / NA / sgRNA / no / [S12]
Salmonella typhymurium / Type I-E
(no Cas3) / 12c / 2 / NA / NA / no / [S25]

a These are only the elements required for the CRISPR-Cas-based system and excludes reporter plasmids etc.

b This represents reduction of the measuredfluorescent, coloured or luminescentproduct rather than gene expression.

c Longer induction times resulted in stronger repression, but these data were not shown.

SUPPLEMENTAL REFERENCES

S1 Jiang, W., et al. (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotech. 31, 233-239

S2 Li, Y., et al. (2015) Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome editing. Metab. Eng. 31, 13-21

S3 Pyne, M.E., et al. (2015) Coupling the CRISPR/Cas9 system with lambda Red recombineering enables simplified chromosomal gene replacement in Escherichia coli. Appl. Environ. Microbiol. 81, 5103-5114

S4 Jiang, Y., et al. (2015) Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl. Environ. Microbiol. 81, 2506-2514

S5 Pines, G., et al. (2015) Codon compression algorithms for saturation mutagenesis. ACS Synth. Biol. 4, 604-614

S6 Reisch, C.R. and Prather, K.L.J. (2015) The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli. Sci. Rep. 5, 15096

S7 Xia, J., et al. (2015) Expression of Shewanella frigidimarina fatty acid metabolic genes in E. coli by CRISPR/Cas9-coupled lambda Red recombineering. Biotechnol. Lett., 1-6

S8 Citorik, R.J., et al. (2014) Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat. Biotech. 32, 1141-1145

S9 Standage-Beier, K., et al. (2015) Targeted large-scale deletion of bacterial genomes using CRISPR-nickases. ACS Synth. Biol. 4, 1217-1225

S10 Wang, Y., et al. (2015) Markerless chromosomal gene deletion in Clostridium beijerinckii using CRISPR/Cas9 system. J. Biotechnol. 200, 1-5

S11 Huang, H., et al. (2015) One-step high-efficiency CRISPR/Cas9-mediated genome editing in Streptomyces. Acta Biochim. Biophys. Sin. 47, 231-243

S12 Tong, Y., et al. (2015) CRISPR-Cas9 based engineering of Actinomycetal genomes. ACS Synth. Biol. 4, 1020-1029

S13 Zeng, H., et al. (2015) Highly efficient editing of the actinorhodin polyketide chain length factor gene in Streptomyces coelicolor M145 using CRISPR/Cas9-CodA(sm) combined system. Appl. Microbiol. Biotechnol. 99, 10575-10585

S14 Cobb, R.E., et al. (2014) High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system. ACS Synth. Biol. 4, 723-728

S15 Oh, J.-H. and van Pijkeren, J.-P. (2014) CRISPR–Cas9-assisted recombineering in Lactobacillus reuteri. Nucleic Acids Res. 42, e131

S16 Xu, T., et al. (2015) Efficient genome editing in Clostridium cellulolyticum via CRISPR-Cas9 nickase. Appl. Environ. Microbiol. 81, 4423-4431

S17 Li, Y., et al. Harnessing Type I and Type III CRISPR-Cas systems for genome editing. Nucleic Acids Res. (in press)

S18 Vercoe, R.B., et al. (2013) Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands. PLoS Genet. 9, e1003454

S19 Gomaa, A.A., et al. (2014) Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems. mBio 5

S20 Bikard, D., et al.(2013) Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 41, 7429-7437

S21 Qi, LeiS., et al. (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173-1183

S22 Ji, W., et al. (2014) Specific gene repression by CRISPRi system transferred through bacterial conjugation. ACS Synth. Biol. 3, 929-931

S23 Lv, L., et al. (2015) Application of CRISPRi for prokaryotic metabolic engineering involving multiple genes, a case study: Controllable P(3HB-co-4HB) biosynthesis. Metab. Eng. 29, 160-168

S24 Luo, M.L., et al. (2015) Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression. Nucleic Acids Res. 43, 674-681

S25 Rath, D., et al. (2015) Efficient programmable gene silencing by Cascade. Nucleic Acids Res. 43, 237-246

S26 Choudhary, E., et al. (2015) Gene silencing by CRISPR interference in mycobacteria. Nat. Commun. 6

S27 Mimee, M., et al. (2015) Programming a human commensal bacterium, Bacteroides thetaiotaomicron, to sense and respond to stimuli in the murine gut microbiota. Cell Syst. 1, 62-71