Rna-guided editing of bacterial genomes using crispr-cas systems

ABSTRACT Here we use the clustered, regularly interspaced, short palindromic repeats (CRISPR)–associated Cas9 endonuclease complexed with dual-RNAs to introduce precise mutations in the

genomes of _Streptococcus pneumoniae_ and _Escherichia coli_. The approach relies on dual-RNA:Cas9-directed cleavage at the targeted genomic site to kill unmutated cells and circumvents the

need for selectable markers or counter-selection systems. We reprogram dual-RNA:Cas9 specificity by changing the sequence of short CRISPR RNA (crRNA) to make single- and multinucleotide

changes carried on editing templates. Simultaneous use of two crRNAs enables multiplex mutagenesis. In _S. pneumoniae_, nearly 100% of cells that were recovered using our approach contained

the desired mutation, and in _E. coli_, 65% that were recovered contained the mutation, when the approach was used in combination with recombineering. We exhaustively analyze dual-RNA:Cas9

target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements, suggesting the versatility of this technique for

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our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS A VERSATILE GENETIC ENGINEERING TOOLKIT FOR _E. COLI_ BASED ON CRISPR-PRIME EDITING Article Open access 01

September 2021 SYSTEMATICALLY ATTENUATING DNA TARGETING ENABLES CRISPR-DRIVEN EDITING IN BACTERIA Article Open access 08 February 2023 MAKE-OR-BREAK PRIME EDITING FOR GENOME ENGINEERING IN

_STREPTOCOCCUS PNEUMONIAE_ Article Open access 23 April 2025 ACCESSION CODES PRIMARY ACCESSIONS NCBI REFERENCE SEQUENCE * KC112384 REFERENCES * Urnov, F.D., Rebar, E.J., Holmes, M.C., Zhang,

H.S. & Gregory, P.D. Genome editing with engineered zinc finger nucleases. _Nat. Rev. Genet._ 11, 636–646 (2010). Article  CAS  PubMed  Google Scholar  * Bogdanove, A.J. & Voytas,

D.F. TAL effectors: customizable proteins for DNA targeting. _Science_ 333, 1843–1846 (2011). Article  CAS  PubMed  Google Scholar  * Stoddard, B.L. Homing endonuclease structure and

function. _Q. Rev. Biophys._ 38, 49–95 (2005). Article  CAS  PubMed  Google Scholar  * Bae, T. & Schneewind, O. Allelic replacement in _Staphylococcus aureus_ with inducible

counter-selection. _Plasmid_ 55, 58–63 (2006). Article  CAS  PubMed  Google Scholar  * Sung, C.K., Li, H., Claverys, J.P. & Morrison, D.A. An _rpsL_ cassette, janus, for gene replacement

through negative selection in _Streptococcus pneumoniae_. _Appl. Environ. Microbiol._ 67, 5190–5196 (2001). Article  CAS  PubMed  PubMed Central  Google Scholar  * Sharan, S.K., Thomason,

L.C., Kuznetsov, S.G. & Court, D.L. Recombineering: a homologous recombination-based method of genetic engineering. _Nat. Protoc._ 4, 206–223 (2009). Article  CAS  PubMed  PubMed Central

  Google Scholar  * Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. _Science_ 337, 816–821 (2012). CAS  PubMed  PubMed Central  Google

Scholar  * Deveau, H., Garneau, J.E. & Moineau, S. CRISPR-Cas system and its role in phage-bacteria interactions. _Annu. Rev. Microbiol._ 64, 475–493 (2010). Article  CAS  PubMed  Google

Scholar  * Horvath, P. & Barrangou, R. CRISPR-Cas, the immune system of bacteria and archaea. _Science_ 327, 167–170 (2010). Article  CAS  PubMed  Google Scholar  * Terns, M.P. &

Terns, R.M. CRISPR-based adaptive immune systems. _Curr. Opin. Microbiol._ 14, 321–327 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * van der Oost, J., Jore, M.M., Westra,

E.R., Lundgren, M. & Brouns, S.J. CRISPR-based adaptive and heritable immunity in prokaryotes. _Trends Biochem. Sci._ 34, 401–407 (2009). Article  CAS  PubMed  Google Scholar  * Brouns,

S.J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. _Science_ 321, 960–964 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * Carte, J., Wang, R., Li, H.,

Terns, R.M. & Terns, M.P. Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. _Genes Dev._ 22, 3489–3496 (2008). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. _Nature_ 471, 602–607 (2011). Article  CAS  PubMed  PubMed Central

  Google Scholar  * Hatoum-Aslan, A., Maniv, I. & Marraffini, L.A. Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism

anchored at the precursor processing site. _Proc. Natl. Acad. Sci. USA_ 108, 21218–21222 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Haurwitz, R.E., Jinek, M.,

Wiedenheft, B., Zhou, K. & Doudna, J.A. Sequence- and structure-specific RNA processing by a CRISPR endonuclease. _Science_ 329, 1355–1358 (2010). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Deveau, H. et al. Phage response to CRISPR-encoded resistance in _Streptococcus thermophilus_. _J. Bacteriol._ 190, 1390–1400 (2008). Article  CAS  PubMed  Google Scholar 

* Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. _Proc. Natl. Acad. Sci.

USA_ 109, E2579–E2586 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Makarova, K.S., Aravind, L., Wolf, Y.I. & Koonin, E.V. Unification of Cas protein families and a

simple scenario for the origin and evolution of CRISPR-Cas systems. _Biol. Direct_ 6, 38 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Barrangou, R. RNA-mediated

programmable DNA cleavage. _Nat. Biotechnol._ 30, 836–838 (2012). Article  CAS  PubMed  Google Scholar  * Brouns, S.J. Molecular biology. A Swiss army knife of immunity. _Science_ 337,

808–809 (2012). Article  CAS  PubMed  Google Scholar  * Carroll, D. A CRISPR approach to gene targeting. _Mol. Ther._ 20, 1658–1660 (2012). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Bikard, D., Hatoum-Aslan, A., Mucida, D. & Marraffini, L.A. CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial

infection. _Cell Host Microbe_ 12, 177–186 (2012). Article  CAS  PubMed  Google Scholar  * Sapranauskas, R. et al. The _Streptococcus thermophilus_ CRISPR-Cas system provides immunity in

_Escherichia coli_. _Nucleic Acids Res._ 39, 9275–9282 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Semenova, E. et al. Interference by clustered regularly interspaced

short palindromic repeat (CRISPR) RNA is governed by a seed sequence. _Proc. Natl. Acad. Sci. USA_ 108, 10098–10103 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Wiedenheft, B. et al. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. _Proc. Natl. Acad. Sci. USA_ 108, 10092–10097 (2011).

Article  CAS  PubMed  PubMed Central  Google Scholar  * Zahner, D. & Hakenbeck, R. The _Streptococcus pneumoniae_ beta-galactosidase is a surface protein. _J. Bacteriol._ 182, 5919–5921

(2000). Article  CAS  PubMed  PubMed Central  Google Scholar  * Marraffini, L.A., Dedent, A.C. & Schneewind, O. Sortases and the art of anchoring proteins to the envelopes of

gram-positive bacteria. _Microbiol. Mol. Biol. Rev._ 70, 192–221 (2006). Article  CAS  PubMed  PubMed Central  Google Scholar  * Motamedi, M.R., Szigety, S.K. & Rosenberg, S.M.

Double-strand-break repair recombination in _Escherichia coli_: physical evidence for a DNA replication mechanism _in vivo_. _Genes Dev._ 13, 2889–2903 (1999). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Hosaka, T. et al. The novel mutation K87E in ribosomal protein S12 enhances protein synthesis activity during the late growth phase in _Escherichia coli_. _Mol.

Genet. Genomics_ 271, 317–324 (2004). Article  CAS  PubMed  Google Scholar  * Costantino, N. & Court, D.L. Enhanced levels of lambda Red-mediated recombinants in mismatch repair mutants.

_Proc. Natl. Acad. Sci. USA_ 100, 15748–15753 (2003). Article  CAS  PubMed  PubMed Central  Google Scholar  * Edgar, R. & Qimron, U. The _Escherichia coli_ CRISPR system protects from

lambda lysogenization, lysogens, and prophage induction. _J. Bacteriol._ 192, 6291–6294 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Marraffini, L.A. & Sontheimer,

E.J. Self versus non-self discrimination during CRISPR RNA-directed immunity. _Nature_ 463, 568–571 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Fischer, S. et al. An

archaeal immune system can detect multiple Protospacer Adjacent Motifs (PAMs) to target invader DNA. _J. Biol. Chem._ 287, 33351–33363 (2012). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Gudbergsdottir, S. et al. Dynamic properties of the _Sulfolobus_ CRISPR-Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers.

_Mol. Microbiol._ 79, 35–49 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Wang, H.H. et al. Genome-scale promoter engineering by coselection MAGE. _Nat. Methods_ 9, 591–593

(2012). Article  PubMed  PubMed Central  Google Scholar  * Cong, L. et al. Multiplex genome engineering using CRISPR-Cas systems. _Science_ doi:10.1126/science.1231143 (3 January 2013). *

Mali, P. et al. RNA-guided human genome engineering via Cas9. _Science_ doi:10.1126/science.1232033 (3 January 2013). * Cho, S.W., Kim, S., Kim, J.M. & Kim, J.-S. Targeted genome

engineering in human cells with the Cas9 RNA-guided endonuclease. _Nat. Biotechnol._ advance online publication, doi:10.1038/nbt.2507 (29 January 2013). * Hwang, W.Y. et al. Efficient genome

editing in zebrafish using a CRISPR-Cas system. _Nat. Biotechnol._ advance online publication, doi:10.1038/nbt.2501 (29 January 2013). * Hoskins, J. et al. Genome of the bacterium

_Streptococcus pneumoniae_ strain R6. _J. Bacteriol._ 183, 5709–5717 (2001). Article  CAS  PubMed  PubMed Central  Google Scholar  * Havarstein, L.S., Coomaraswamy, G. & Morrison, D.A.

An unmodified heptadecapeptide pheromone induces competence for genetic transformation in _Streptococcus pneumoniae_. _Proc. Natl. Acad. Sci. USA_ 92, 11140–11144 (1995). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Horinouchi, S. & Weisblum, B. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance.

_J. Bacteriol._ 150, 815–825 (1982). CAS  PubMed  PubMed Central  Google Scholar  * Horton, R.M. In vitro recombination and mutagenesis of DNA: SOEing Together tailor-made genes. _Methods

Mol. Biol._ 15, 251–261 (1993). CAS  PubMed  Google Scholar  * Podbielski, A., Spellerberg, B., Woischnik, M., Pohl, B. & Lutticken, R. Novel series of plasmid vectors for gene

inactivation and expression analysis in group A streptococci (GAS). _Gene_ 177, 137–147 (1996). Article  CAS  PubMed  Google Scholar  * Husmann, L.K., Scott, J.R., Lindahl, G. &

Stenberg, L. Expression of the Arp protein, a member of the M protein family, is not sufficient to inhibit phagocytosis of _Streptococcus pyogenes_. _Infect. Immun._ 63, 345–348 (1995). CAS

  PubMed  PubMed Central  Google Scholar  * Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. _Nat. Methods_ 6, 343–345 (2009). Article  CAS  PubMed 

Google Scholar  Download references ACKNOWLEDGEMENTS We thank V. Fischetti and C. Euler for plasmid pLZ12spec, D. Court for the HME63 strain, J. Kern for plasmid pKD46, the Rockefeller

University Genomic Resource Center for technical support and J. Roberts for the MG1655 strain. D.B. is supported by a Harvey L. Karp Discovery Award and the Bettencourt Schuller Foundation.

D.C. is supported by the Medical Scientist Training Program. F.Z. is supported by a US National Institutes of Health (NIH) Director's Pioneer Award (DP1MH100706), Transformative R01,

the Keck, McKnight, Gates, Damon Runyon, Searle Scholars, Klingenstein, and Simons Foundations, Bob Metcalfe, Mike Boylan and Jane Pauley. L.A.M. is supported by the Searle Scholars Program,

the Rita Allen Scholars Program, a Irma T. Hirschl Award and a NIH Director's New Innovator Award (1DP2AI104556-01). AUTHOR INFORMATION Author notes * Wenyan Jiang and David Bikard:

These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Laboratory of Bacteriology, The Rockefeller University, New York, New York, USA Wenyan Jiang, David Bikard & 

Luciano A Marraffini * Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA David Cox & Feng Zhang * Department of Brain and Cognitive Science and Department of Biological

Engineering, McGovern Institute for Brain Research, MIT, Cambridge, Massachusetts, USA David Cox & Feng Zhang Authors * Wenyan Jiang View author publications You can also search for this

author inPubMed Google Scholar * David Bikard View author publications You can also search for this author inPubMed Google Scholar * David Cox View author publications You can also search

for this author inPubMed Google Scholar * Feng Zhang View author publications You can also search for this author inPubMed Google Scholar * Luciano A Marraffini View author publications You

can also search for this author inPubMed Google Scholar CONTRIBUTIONS W.J., D.B. and L.A.M. designed the experiments; W.J., D.B. and D.C. performed experiments; W.J., D.B., F.Z. and L.A.M.

wrote the paper. CORRESPONDING AUTHORS Correspondence to David Bikard or Luciano A Marraffini. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests.

SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary Discussion, Supplementary Figures 1–11 and Supplementary Tables 1–3 (PDF 2790 kb) SUPPLEMENTARY DATA Supplementary Data

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CRISPR-Cas systems. _Nat Biotechnol_ 31, 233–239 (2013). https://doi.org/10.1038/nbt.2508 Download citation * Received: 29 November 2012 * Accepted: 16 January 2013 * Published: 29 January

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