CRISPR Methods for Bacterial Genome Engineering

Posted by Mary Gearing on Mar 3, 2016 10:30:00 AM


This post was updated on Dec 5, 2017.

Although CRISPR systems were first discovered in bacteria, most CRISPR-based genome engineering has taken place in other organisms. In many bacteria, unlike other organisms, CRISPR-induced double stranded breaks are lethal because the non-homologous end-joining (NHEJ) repair pathway is not very robust. In many cases, homology-directed repair does not function effectively either, but scientists have devised means of co-opting phage genetic systems to facilitate homologous recombination in bacteria. These quirks change the way CRISPR-mediated genome engineering functions in bacteria, but have no fear - plasmids from Addgene depositors are making it easier than ever to do CRISPR editing in E. coli and other commonly-used bacterial species. Read on to learn about the tools available for bacteria and some of the applications for which they’ve been used.Click to Download Addgene's CRISPR 101 eBook

The Beginnings of Bacterial CRISPR Engineering

Much bacterial genome engineering is done with recombineering, a technique that utilizes phage recombination machinery to promote homologous recombination of linear DNA fragments. Since recombineering does not contain a selection step for successful modifications, efficiency can be low, especially for larger modifications.

What’s the solution to this inefficiency? Use CRISPR to make it a selectable process! As NHEJ is ineffective in bacteria, CRISPR-induced double stranded breaks (DSB) are lethal. Addgene depositor Luciano Marraffini’s lab took advantage of this lethality to design the first synthetic bacterial CRISPR system in E. coli. The system available from Addgene consists of two plasmids:

  1. pCas9: carries Cas9 and chloramphenicol resistance
  2. pCRISPR: carries a spacer targeting the gene of interest and kanamycin resistance

E. coli carrying the phage recombineering machinery are first electroporated with pCas9. Then, pCRISPR is introduced along with an oligonucleotide repair template. Through recombineering, the locus of interest is modified to match the repair template, and the locus cannot be recognized by the spacer-derived crRNA. However, if recombineering is unsuccessful and the wild-type sequence persists, Cas9 will cleave the gene of interest, inducing a lethal DSB.

Bacterial_CRISPR_Schematic.png

This system is distinct from those used in eukaryotes in that CRISPR isn’t the primary editing force; in contrast, in E. coli, CRISPR is primarily a means of selection that targets cells in which homologous recombination has not occurred. This powerful negative selection system ensures high editing efficiency; the only non-edited cells to survive have inactivating mutations in the Cas9 or spacer sequence, and these rare events are easily detectable using PCR. The system designed by Jiang et al. 2013 also functions in S. pneumoniae and can be used to generate multiple mutations simultaneously.

What's New: CRISPR Multiplexing in E. coli and Other Bacteria

CRISPR is clearly a powerful tool for bacterial engineering, and the work needed to adapt these systems to cover most bacterial species is ongoing. The good news is that CRISPR multiplexing is now available for multiple bacterial species/genera. See below for a list of plasmid-based systems available from Addgene - we hope your favorite bacteria are included!

Genome Editing

Nielsen Lab E. coli CRMAGE Plasmids:

Ronda et al.'s CRMAGE system is a fast, multiplexable method that combines CRISPR and recombineering-based MAGE (Multiplex Automated Genome Engineering) technology. pMA7CR_2.0 expresses lambda Red and Cas9, which are separately inducible by L-arabinose and anhydrotetracycline (aTet), respectively. pMAZ-SK contains an aTet-inducible gRNA and a backbone-targeting gRNA cassette for plasmid curing through "self-destruction" after induction with L-rhamnose and aTet. CRMAGE is much more efficient than traditional recombineering, with 96-99% efficiency for point mutations and 66% efficiency for small insertions. Multiplexing of two targets simultaneously is possible with efficiency >70%. CRMAGE is an incredibly fast protocol, with only 5 hours incubation time needed for a single round of editing, and a subsequent curing protocol that requires only 2-3 hours incubation. 

Sheng Yang Lab E. coli and T. citrea Scarless Editing Plasmids:

Jiang et al. 2015 describes a two-plasmid system that combines recombineering with CRISPR to create a system for scarless, iterative genome engineering. pCas contains Cas9 and the phage recombination gene lambda Red. pTargetF contains the specific gRNA(s), and the repair template is supplied as a dsDNA fragment. Gene deletion efficiency is as high as ~69%, but insertion efficiency varies with the length of homology supplied with the template (40 bp - 6% vs 400 bp - 28%.) Each round of editing takes two days, and the pTargetF and pCas plasmids can be cured from the bacteria via non-selection and growth at 37 °C, respectively. Although developed in E. coli, the system was used successfully in Tatumella citrea, another species of Enterobacteriaceae, without the need for modification. This finding suggests that the system may be functional in most Enterobacteriaceae.

Prather Lab noSCAR E. coli Plasmids:

Like Jiang et al.’s system, Reisch and Prather’s noSCAR system incorporates phage recombination machinery into CRISPR editing to create scarless modifications. This two-plasmid system uses ssDNA or dsDNA repair templates to produce point mutations, insertions, or deletions. Tet-inducible Cas9 is located on the pCas9cr4 plasmid, and the targeting gRNA and recombination machinery are carried by pKDsg-xxx. pKDsg-xxx is easily cured after the desired modification has been made, allowing for multiple sequential rounds of editing. For most of the experiments conducted, 100% of colonies tested via colony PCR or sequencing displayed the desired mutation, indicating that the noSCAR method is highly efficient.

Tao Chen Lab E. coli Iterative Editing Plasmids:

Li et al.’s two-plasmid system enables easy metabolic engineering in E. coli carrying phage recombination machinery. pGRB supplies one or more gRNAs inserted using Golden Gate Assembly. Cas9cur contains Cas9 and an inducible gRNA targeting the bla resistance gene, which can be used to rapidly cure the bla-containing pGRB. Either ssDNA or dsDNA may be used as a repair template. Each cycle of genome modification takes two days, and the system displays ~100% modification efficiency for deletions as large as 12 kb and insertions as large as 2 kb. Multiple mutations can be introduced simultaneously, albeit at a lower efficiency (83% for 2 mutations and 23% for 3 mutations.)

Huimin Zhao Lab pCRISPomyces Plasmids:

Streptomyces bacteria produce a wide variety of bioactive natural products. To easily explore and engineer pathways within this genus, Cobb et al. created two “pCRISPomyces” systems for use in Streptomyces. pCRISPomyces-1 includes Cas9, a tracrRNA, and a CRISPR array, while pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of pCRISPomyces-2 displays a higher editing efficiency, perhaps due to its condensed design. For both systems, custom spacers/gRNAs are easily inserted using BbsI and Golden Gate Assembly. Either plasmid can be also linearized with XbaI to insert extra elements, like a repair template, using Gibson Assembly or restriction enzyme cloning. Streptomyces bacteria are more recombinogenic than E. coli, so this system functions more similarly to CRISPR/Cas9 systems adapted for eukaryotes in that Cas9-mediated cleavage induces HDR to directly modify a given gene.

Sheng Yang Lab Cpf1 C. glutamicum Plasmids:

Jiang et al. 2017 found that SpCas9 was toxic in C. glutamicum, a difficult-to-engineer bacterium. They created an editing system based on FnCpf1 that also uses ssDNA recombineering. For small genomic alterations, FnCpf1 is expressed along with inducible E.coli recT from pJYS1Ptac or pJYS1Peftu and the target crRNA is supplied on a pJYS2 series plasmid. This two-plasmid system displays an editing efficiency of about 80-100% for small genomic changes, with iterative editing taking place in 3N+4 days, where N is the rounds of editing. The all-in-one plasmid pJYS3_ΔcrtYf, which contains FnCpf1, recT, and homology arms, can be used for larger deletions and insertions in 3N+2 days at 5-15% efficiency.

Transcriptional Repression

Bacterial CRISPR methods are also available for transcriptional activation and repression. As RNA interference does not function in bacteria, most previous efforts to regulate gene expression were limited to the use of inducible promoters or direct gene knockout. In contrast, CRISPR offers a much more user-friendly way to modulate gene expression. Both Bikard et al. and Qi et al. developed early systems for use in E. coli; while Bikard et al. used a native minimal CRISPR array, Qi et al. employed a gRNA-based design more familiar to those using CRISPR in eukaryotes. As in other systems, catalytically dead (dCas9) targeted to a promoter or gene body can repress transcription by physically blocking the elongation complex, and gRNAs targeting the noncoding strand repress transcription more efficiently than those targeting the coding strand. Bikard et al. also successfully activated transcription in E. coli and S. pneumoniae using a Cas9-RNA polymerase omega subunit fusion guided to bind 80-100 bases upstream of the transcription start site.

Koffas Lab CRISPathBrick Multiplex Plasmid:

This system allows you to assemble type II-A CRISPR arrays for dCas9-based transcriptional repression in E. coli. The pCRISPathBrick plasmid contains dCas9 and a nontargeting spacer flanked by two CRISPR repeats. The spacer can be digested using BsaI, allowing a spacer-repeat “brick” to be inserted. The BsaI site remains intact, allowing subsequent “bricks” to be added one by one. This approach is especially useful for combinatorial analyses. For example, if you were to develop an array using 3 distinct spacer-repeats (more are possible), you could easily create 7 unique arrays (e.g. for spacers A, B, and C, you could obtain arrays A, B, C, AB, AC, BC, and ABC).

Beisel Lab Type I CRISPR Plasmids:

Luo et al. took a different approach to transcriptional repression: instead of adding a Type II system for transcriptional repression, they co-opted a native Type I system in E. coli. Whereas Type II systems require a single protein for DNA cleavage, Type I systems employ a multi-Cas protein complex. Luo et al. deleted one component of the Type I complex, the nuclease cas3, from the E. coli genome in order to create an inactive complex. Without Cas3 present, the other Type I Cas proteins can tightly bind a given locus and block transcription. pcrRNA.con and pcrRNA.ind are constitutive and arabinose-inducible empty array plasmids into which desired spacers can be cloned. Using an endogenous Type I system instead of the common Type II system decreases the amount of genetic material that must be transformed into the bacteria, and opens up more potential PAM sites (in this case, CTT and CCT).

Timothy Lu Lab C. glutamicum CRISPRi Plasmids:

Cleto et al. developed a two-plasmid system for CRISPRi in C. glutamicum. IPTG-inducible dCas9 and a gRNA are expressed from pZ8-T_dCas9 and pAL, respectively. This system decreased target gene mRNA levels by 84-98%. They also found that targeting the non-template strand caused more efficient repression than targeting the template strand.

Robert Husson Lab M. tuberculosis CRISPRi Plasmids:

Singh et al. used CRISPRi to study essential gene in M. tuberculosis. dCas9 and a gRNA are expressed on pRH2502 and pRH2521, respectively. Both dCas9 and the gRNA are Tet-inducible, and a gRNA can be cloned easily into pRH2521 using BbsI. Singh et al. achieved 80-90% RNA knockdown across multiple gRNAs per gene.


References

1. Jiang, Wenyan, et al. (2013). “RNA-guided editing of bacterial genomes using CRISPR-Cas systems.” Nat Biotechnol. 31(3): 233-9. PubMed PMID: 23360965. PubMed Central PMCID: PMC3748948.

2. Bikard, David, et al. (2013). “Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system.” Nucleic Acids Res. 41(15): 4729-37. PubMed PMID: 23761437. PubMed Central PMCID: PMC3753641.
3. Qi, Lei. S., et al. (2013). “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.” Cell 152(5): 1173-83. PubMed PMID: 23452860. PubMed Central PMCID: PMC3664290.
4. Cobb, Ryan E., Wang, Yajie, and Huimin Zhao. (2015). “High-Efficiency Multiplex Genome Editing of Streptomyces Species Using an Engineered CRISPR/Cas System.” ACS Synth Biol. 4(6): 723-8. PubMed PMID: 25458909. PubMed Central PMCID: PMC4459934.

5. Ronda, Carlotta, et al. (2016). "CRMAGE: CRISPR Optimized MAGE Recombineering." Sci Rep. 6:19452. PubMed PMID: 26797514

6. Jiang, Yu, et al. (2015). “Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol. 81(7): 2506-14. PubMed PMID: 25636838. PubMed Central PMCID: PMC4357945.

7. Reisch, Chris R. and Kristala L. J. Prather. “The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli.” Sci Rep. 5: 15096. PubMed PMID: 26463009. PubMed Central PMCID: PMC4604488.
8. Li, Yifan, et al. (2015). “Metabolic engineering of Escherichia coli using CRISPR-Cas mediated genome editing.” Metab Eng. 31:13-21. PubMed PMID: 26141150.

9. Jiang, Yu, et al. (2017). "CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum." Nat Commun. 8:15179. PubMed PMID: 28469274

10. Cress, Brady F., et al. (2015). “CRISPathBrick: Modular Combinatorial Assembly of Type II-A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli.ACS Synth Biol. 4(9): 987-1000. PubMed PMID:25822415.

11. Luo, Michelle L., Mullis, Adam S., Leenay, Ryan T. and Chase L. Beisel. (2015). “Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression.” Nucleic Acids Res. 43(1): 674-681. PubMed PMID: 25326321. PubMed Central PMCID: PMC4288209.

12. Cleto, Sara, et al. (2016). "Corynebacterium glutamicum Metabolic Engineering with CRISPR Interference (CRISPRi)." ACS Synth Biol. 5(5): 375–385. PubMed PMID: 26829286

13. Singh, Atul K., et al. (2016). "Investigating essential gene function in Mycobacterium tuberculosis using an efficient CRISPR interference system." Nucleic Acids Res. 44(18):e143. PubMed PMID: 27407107

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