A Cas12a-based CRISPR interference system for multigene regulation in mycobacteria

Abstract

Mycobacteria are responsible for a heavy global disease burden, but their relative genetic intractability has long frustrated research efforts. The introduction of clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) has made gene repression in mycobacteria much more efficient, but limitations of the prototypical Cas9-based platform, for example, in multigene regulation, remain. Here, we introduce an alternative CRISPRi platform for mycobacteria that is based on the minimal type V Cas12a enzyme in combination with synthetic CRISPR arrays. This system is simple, tunable, reversible, can efficiently regulate essential genes and multiple genes simultaneously, and works as efficiently in infected macrophages as it does in vitro. Together, Cas12a-based CRISPRi provides a facile tool to probe higher-order genetic interactions in mycobacteria including Mycobacterium tuberculosis (Mtb), which will enable the development of synthetically lethal drug targets and the study of genes conditionally essential during infection. Mycobacteria are responsible for a heavy global disease burden, but their relative genetic intractability has long frustrated research efforts. The introduction of clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) has made gene repression in mycobacteria much more efficient, but limitations of the prototypical Cas9-based platform, for example, in multigene regulation, remain. Here, we introduce an alternative CRISPRi platform for mycobacteria that is based on the minimal type V Cas12a enzyme in combination with synthetic CRISPR arrays. This system is simple, tunable, reversible, can efficiently regulate essential genes and multiple genes simultaneously, and works as efficiently in infected macrophages as it does in vitro. Together, Cas12a-based CRISPRi provides a facile tool to probe higher-order genetic interactions in mycobacteria including Mycobacterium tuberculosis (Mtb), which will enable the development of synthetically lethal drug targets and the study of genes conditionally essential during infection. The adaptive bacterial immune systems based on clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have transformed genetic manipulation, and the ease with which they can be programmed has led to their wide use in gene editing in eukaryotes and prokaryotes (1Knott G.J. Doudna J.A. CRISPR-Cas guides the future of genetic engineering.Science. 2018; 361: 866-869Crossref PubMed Scopus (500) Google Scholar). One application of the CRISPR/Cas system, CRISPR interference (CRISPRi), introduced a new way of gene regulation by coexpressing an inactive Cas9 nuclease with an engineered single guide RNA (sgRNA) that directs the inactive nuclease to a target gene where it blocks transcription rather than cleaves the DNA (2Qi L.S. Larson M.H. Gilbert L.A. Doudna J.A. Weissman J.S. Arkin A.P. Lim W.A. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.Cell. 2013; 152: 1173-1183Abstract Full Text Full Text PDF PubMed Scopus (2538) Google Scholar). The prototypical CRISPRi system is based on an inactive type 2-II Cas9 nuclease (dCas9) and has recently also been adapted for use in mycobacteria including Mtb (3Choudhary E. Thakur P. Pareek M. Agarwal N. Gene silencing by CRISPR interference in mycobacteria.Nat. Commun. 2015; 6: 6267Crossref PubMed Scopus (132) Google Scholar, 4Rock J.M. Hopkins F.F. Chavez A. Diallo M. Chase M.R. Gerrick E.R. Pritchard J.R. Church G.M. Rubin E.J. Sassetti C.M. Schnappinger D. Fortune S.M. Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform.Nat. Microbiol. 2017; 2: 16274Crossref PubMed Scopus (163) Google Scholar, 5Singh A.K. Carette X. Potluri L.P. Sharp J.D. Xu R. Prisic S. Husson R.N. Investigating essential gene function in Mycobacterium tuberculosis using an efficient CRISPR interference system.Nucleic Acids Res. 2016; 44e143Crossref PubMed Scopus (66) Google Scholar), for which genetic manipulation has long been an experimental bottleneck. Genetic manipulation of Mtb poses many challenges. The slow growth of Mtb makes all manipulations requiring chromosomal changes time-consuming, and while the high rate of illegitimate recombination in Mtb has been overcome by expressing heterologous recombineering enzymes (6van Kessel J.C. Hatfull G.F. Recombineering in Mycobacterium tuberculosis.Nat. Methods. 2007; 4: 147-152Crossref PubMed Scopus (338) Google Scholar), allelic exchange and possibly removal of selection markers can still take weeks to months. Tunable gene repression and repression of essential genes before the introduction of CRISPRi required the introduction of regulatable promoters such as the tetracycline- or pristinamycin-inducible promoters (7Ehrt S. Schnappinger D. Controlling gene expression in mycobacteria.Future Microbiol. 2006; 1: 177-184Crossref PubMed Scopus (23) Google Scholar) or sequences for regulated protein degradation (8Wei J.R. Krishnamoorthy V. Murphy K. Kim J.H. Schnappinger D. Alber T. Sassetti C.M. Rhee K.Y. Rubin E.J. Depletion of antibiotic targets has widely varying effects on growth.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 4176-4181Crossref PubMed Scopus (115) Google Scholar) in the chromosomal copy of a target gene. These approaches had varying efficiency, and leaky expression or repression could lead to incomplete control of target genes. While these challenges already complicated the manipulation of single genes, they multiplied for the manipulation of multiple genes at once. As a result, our understanding of genetic interactions in Mtb is lagging behind that in other bacteria. For genome-wide studies in Mtb, transposon mutagenesis has become a powerful tool, but insertion of transposons produces libraries with undefined mutations that require sequencing for deconvolution. Also, transposon libraries do not include essential genes, the genes that are arguably the most relevant, for example, in drug discovery. CRISPRi genome-wide libraries have been developed for eukaryotes and have been used, for example, for probing the host genetic factors for Mtb infection (9Lai Y. Babunovic G.H. Cui L. Dedon P.C. Doench J.G. Fortune S.M. Lu T.K. Illuminating host-mycobacterial interactions with genome-wide CRISPR knockout and CRISPRi screens.Cell Syst. 2020; 11: 239-251.e237Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). With their defined nature, tunability, and inclusion of essential genes, CRISPRi Mtb mutant libraries seem poised for global approaches to understand Mtb gene function. Despite the advantages of CRISPRi, however, knockdown efficiency can vary widely for different target genes, and simultaneous manipulation of more than one gene remains challenging. These limitations have hampered attempts to probe redundant genes, gene families, and higher-order genetic interactions. Natural CRISPR systems are inherently multigene regulatory systems that can principally also be reprogrammed to regulate multiple genes at once. One current limitation of the Cas9-based system, however, is the relatively large size of the sgRNA which requires a crRNA and a tracrRNA that are typically fused to produce the >100 bp long sgRNA. Expression of multiple sgRNAs for multigene knockdown requires stepwise cloning of large individual transcriptional units for each sgRNA and in mycobacteria has shown mostly moderate knockdown efficiency of 2- to 3-fold for most genes (10Agarwal N. Construction of a novel CRISPRi-based tool for silencing of multiple genes in Mycobacterium tuberculosis.Plasmid. 2020; 110: 102515Crossref PubMed Scopus (2) Google Scholar). The natural diversity of CRISPR systems, however, may offer simpler solutions to CRISPRi in Mtb: Recently, the minimal type 2-V CRISPR enzyme Cas12a (previously Cpf1) has been described (11Zetsche B. Gootenberg J.S. Abudayyeh O.O. Slaymaker I.M. Makarova K.S. Essletzbichler P. Volz S.E. Joung J. van der Oost J. Regev A. Koonin E.V. Zhang F. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.Cell. 2015; 163: 759-771Abstract Full Text Full Text PDF PubMed Scopus (1983) Google Scholar). Cas12a does not require a tracrRNA and combines pre-crRNA processing and interference functions in one enzyme (12Fonfara I. Richter H. Bratovic M. Le Rhun A. Charpentier E. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA.Nature. 2016; 532: 517-521Crossref PubMed Scopus (436) Google Scholar). The combination of these biochemical functions makes Cas12a a stand-alone enzyme that at least in some bacteria only requires a synthetic CRISPR array for the generation of mature crRNAs. This simplified system has been rapidly adapted for multiplex gene editing in mammalian cells (13Zetsche B. Heidenreich M. Mohanraju P. Fedorova I. Kneppers J. DeGennaro E.M. Winblad N. Choudhury S.R. Abudayyeh O.O. Gootenberg J.S. Wu W.Y. Scott D.A. Severinov K. van der Oost J. Zhang F. Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array.Nat. Biotechnol. 2017; 35: 31-34Crossref PubMed Scopus (421) Google Scholar), plants (14Wang M. Mao Y. Lu Y. Tao X. Zhu J.K. Multiplex gene editing in rice using the CRISPR-Cpf1 system.Mol. Plant. 2017; 10: 1011-1013Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), and bacteria (15Li L. Wei K. Zheng G. Liu X. Chen S. Jiang W. Lu Y. CRISPR-Cpf1-assisted multiplex genome editing and transcriptional repression in Streptomyces.Appl. Environ. Microbiol. 2018; 84e00827-18Crossref PubMed Scopus (64) Google Scholar) and has recently also been exploited for CRISPRi in Streptomyces (15Li L. Wei K. Zheng G. Liu X. Chen S. Jiang W. Lu Y. CRISPR-Cpf1-assisted multiplex genome editing and transcriptional repression in Streptomyces.Appl. Environ. Microbiol. 2018; 84e00827-18Crossref PubMed Scopus (64) Google Scholar), E. coli (16Zhang X. Wang J. Cheng Q. Zheng X. Zhao G. Wang J. Multiplex gene regulation by CRISPR-ddCpf1.Cell Discov. 2017; 3: 17018Crossref PubMed Scopus (93) Google Scholar), and the cyanobacterium Synechococcus (17Choi S.Y. Woo H.M. CRISPRi-dCas12a: A dCas12a-mediated CRISPR interference for repression of multiple genes and metabolic engineering in Cyanobacteria.ACS Synth. Biol. 2020; 9: 2351-2361Crossref PubMed Scopus (16) Google Scholar, 18Knoot C.J. Biswas S. Pakrasi H.B. Tunable repression of key photosynthetic processes using Cas12a CRISPR interference in the fast-growing Cyanobacterium Synechococcus sp. UTEX 2973.ACS Synth. Biol. 2020; 9: 132-143Crossref PubMed Scopus (14) Google Scholar) (Fig. 1). Here, we adapted this system for CRISPRi in mycobacteria, creating a simple, highly tunable, reversible, multigene regulation platform. To test whether dCas12a in conjunction with a synthetic CRISPR array can be used for CRISPRi in mycobacteria, we stably introduced the gene for the inactive Francisella novicida Cas12a mutant Asp917Ala (dCas12a) into the Tweety recombination site of Mycobacterium smegmatis (Msm) strain mc2155 (19Pham T.T. Jacobs-Sera D. Pedulla M.L. Hendrix R.W. Hatfull G.F. Comparative genomic analysis of mycobacteriophage tweety: Evolutionary insights and construction of compatible site-specific integration vectors for mycobacteria.Microbiology (Reading). 2007; 153: 2711-2723Crossref PubMed Scopus (61) Google Scholar). The Asp917Ala mutant abrogates DNA cleavage activity but retains pre-CRISPR RNA processing activity (12Fonfara I. Richter H. Bratovic M. Le Rhun A. Charpentier E. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA.Nature. 2016; 532: 517-521Crossref PubMed Scopus (436) Google Scholar). Initially, we could not detect the Francisella-derived dCas12a enzyme in Msm by Western expression a The Francisella is while are Msm To test whether these we a Francisella gene that for expression and has a of This in Msm and to test whether Msm expressing dCas12a can synthetic CRISPR into and transcription of target genes. the strain by an into the Tweety recombination site and a which in mycobacteria N. A. T. J. T. G.J. U. S. of for use with PubMed Scopus Google Scholar), into the recombination the of the by expressing synthetic CRISPR arrays. The CRISPR the Francisella each bp to the and (Fig. target on to be the or that Francisella Cas12a to the target DNA (11Zetsche B. Gootenberg J.S. Abudayyeh O.O. Slaymaker I.M. Makarova K.S. Essletzbichler P. Volz S.E. Joung J. van der Oost J. Regev A. Koonin E.V. Zhang F. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.Cell. 2015; 163: 759-771Abstract Full Text Full Text PDF PubMed Scopus (1983) Google Scholar, Zhao H. L. the key of the Cpf1 for transcription regulation in Syst. Biotechnol. 4: PubMed Scopus Google Scholar). The synthetic CRISPR for of pre-crRNA an expression the control of in the we activity for As we a strain expressing a array bp sequences that to Msm or Mtb sequences and in each strain with and of in all dCas12a with with the and the in on the of the with one to with and that may be for knockdown to or to To test the of dCas12a-mediated knockdown in mycobacteria, we the of the and sequences in and with a of the expression of genes, but to much than the of the (Fig. with that the is the target for Francisella Cas12a (16Zhang X. Wang J. Cheng Q. Zheng X. Zhao G. Wang J. Multiplex gene regulation by CRISPR-ddCpf1.Cell Discov. 2017; 3: 17018Crossref PubMed Scopus (93) Google Scholar, Zhao H. L. the key of the Cpf1 for transcription regulation in Syst. Biotechnol. 4: PubMed Scopus Google Scholar). of Msm by expression of dCas12a and the (Fig. To test whether the use of multiple a single has an on we the array with each of (Fig. The individual with efficiency in the in which they to the and their in than the of the individual moderate we dCas12a a we knockdown of in the of array by not that of leaky expression of the array can lead to expressing the array and dCas12a promoters for a of and used for this To the of we the of a of on (Fig. of as as and These that can pre-crRNA into mature and genes in the of to the simultaneous repression of genes, the for all genes in these could not single or multigene To test for multigene knockdown of genes, we a strain of Msm that a synthetic CRISPR array essential genes that are required for the of the and introduced for each gene into a single CRISPR of the array in Msm expressing the strain growth in (Fig. by the growth to type with one of the not growth (Fig. These that genes and that dCas12a can target multiple genes as as essential genes. whether dCas12a-mediated CRISPRi can be adapted to introduced synthetic CRISPR the into an Mtb strain dCas12a in the Tweety recombination and the in the recombination of the with in efficient of (Fig. The of knockdown on the of the with one in and and in of to for knockdown and knockdown to to the with not the Expression of dCas12a and the not have an on growth of Mtb (Fig. To test the of the system, we the Mtb strain an array with the for and the by and in to the the activity (Fig. These that the system is highly and can be by the To test the of and we global gene expression in the and the array by RNA While expression changes with effects of the a of genes of these genes had to the and their may be a to the of by in the strain (Fig. we to test for multigene regulation in an array the genes and with for each To detect knockdown of all genes and the efficiency of multigene we the knockdown by genes with and in (Fig. of the gene which not by the that expression of dCas12a and had effects global These that at least genes can be by dCas12a CRISPRi in mycobacteria has only been used to regulate bacterial genes in (3Choudhary E. Thakur P. Pareek M. Agarwal N. Gene silencing by CRISPR interference in mycobacteria.Nat. Commun. 2015; 6: 6267Crossref PubMed Scopus (132) Google Scholar, 4Rock J.M. Hopkins F.F. Chavez A. Diallo M. Chase M.R. Gerrick E.R. Pritchard J.R. Church G.M. Rubin E.J. Sassetti C.M. Schnappinger D. Fortune S.M. Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform.Nat. Microbiol. 2017; 2: 16274Crossref PubMed Scopus (163) Google Scholar, 5Singh A.K. Carette X. Potluri L.P. Sharp J.D. Xu R. Prisic S. Husson R.N. Investigating essential gene function in Mycobacterium tuberculosis using an efficient CRISPR interference system.Nucleic Acids Res. 2016; 44e143Crossref PubMed Scopus (66) Google Scholar). To test whether the system can also be used to regulate gene expression during we infected a used for Mtb infection for the of growth of Mycobacterium tuberculosis PubMed Scopus Google Scholar), with a strain an array with the CRISPRi with or with infection with the CRISPRi strain the array knockdown of to the knockdown in (Fig. To that CRISPRi led to which Mtb and the we with the gene repression had on that the in to gene repression (Fig. The introduction of CRISPRi to the of has many of the Cas9-based system has some and in multigene knockdown remains challenging. Here, we exploited the multigene of CRISPR systems, in the minimal to more efficient multigene regulation in the for the of genes that can be with this system remains to be it is than the To genes in our we used that could also be at different gene the efficiency of individual The natural Francisella CRISPR to the natural for the The of knockdown by CRISPRi can be by varying the of the by the of and by using target sequences with different The are and require our system, knockdown efficiency can be by varying the of each a more as each produces of an alternative CRISPRi system provides for genes that may be to target in other For multigene we a the efficiency of knockdown and the of genes that be for array The of the sequences of the synthetic also challenges for gene we could with as many as For facile multigene regulation, Cas12a has advantages Cas9-based CRISPRi The much and sequences bp and and the single transcriptional required for of the Cas12a-based system more than the current Cas9-based system, which requires of >100 with individual and of Cas12a-based CRISPRi is genome The genome of CRISPRi is by the for a enzyme and is a for a CRISPRi The Cas12a of the Mtb genome is more than that of S. the most efficient Cas9 used in mycobacteria J.M. Hopkins F.F. Chavez A. Diallo M. Chase M.R. Gerrick E.R. Pritchard J.R. Church G.M. Rubin E.J. Sassetti C.M. Schnappinger D. Fortune S.M. Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform.Nat. Microbiol. 2017; 2: 16274Crossref PubMed Scopus (163) Google Scholar). The for S. is in of Mtb the Cas12a is in of Mtb sequences and targets on with efficient for enzymes J.M. Hopkins F.F. Chavez A. Diallo M. Chase M.R. Gerrick E.R. Pritchard J.R. Church G.M. Rubin E.J. Sassetti C.M. Schnappinger D. Fortune S.M. Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform.Nat. Microbiol. 2017; 2: 16274Crossref PubMed Scopus (163) Google Scholar, Zhao H. L. the key of the Cpf1 for transcription regulation in Syst. Biotechnol. 4: PubMed Scopus Google Scholar, R.N. R. and diversity 2016; Full Text Full Text PDF PubMed Scopus (163) Google Scholar), S. Cas9 can target the Mtb genome on for a Cas12a and can target of genes. the S. Cas9 a that is more the enzyme is not as efficient in gene repression in mycobacteria (3Choudhary E. Thakur P. Pareek M. Agarwal N. Gene silencing by CRISPR interference in mycobacteria.Nat. Commun. 2015; 6: 6267Crossref PubMed Scopus (132) Google Scholar, 4Rock J.M. Hopkins F.F. Chavez A. Diallo M. Chase M.R. Gerrick E.R. Pritchard J.R. Church G.M. Rubin E.J. Sassetti C.M. Schnappinger D. Fortune S.M. Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform.Nat. Microbiol. 2017; 2: 16274Crossref PubMed Scopus (163) Google Scholar). Cas12a with have recently been described K. J. I. L. Joung J.K. with and for and Biotechnol. PubMed Scopus Google Scholar), for example, an engineered mutant of the Cas12a enzyme L. J.C. T. H. N. Zhang F. Cpf1 with Biotechnol. 2017; 35: PubMed Scopus Google Scholar) that the which could genome While we not the efficiency of Cas9 and Cas12a-based CRISPRi, Cas9-based CRISPRi in Msm by to in one study J.M. Hopkins F.F. Chavez A. Diallo M. Chase M.R. Gerrick E.R. Pritchard J.R. Church G.M. Rubin E.J. Sassetti C.M. Schnappinger D. Fortune S.M. Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform.Nat. Microbiol. 2017; 2: 16274Crossref PubMed Scopus (163) Google Scholar), on the Cas9 used, with in of in our The large the efficiency of Cas9 that other Cas12a may also to the system, our system is for Mtb genes, which has the study of many gene such as the and genes. is a of tuberculosis and such requires of and interactions. With our system, the of interactions multiple genes to synthetic is The CRISPRi system gene expression as efficiently in infected cells as it in which the use to the study of genes conditionally essential during infection and to interactions. the system used for the control of dCas12a and the synthetic is also in we that our system also works in the efficiency of knockdown in and in macrophages will for the probing of for example, interactions Mtb that the of tuberculosis K. H. K. L. in mycobacteria by a 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). Together, we introduce a new CRISPRi system for mycobacteria for and efficient multigene regulation in and in infected This Cas12a-based system is and the efficient multigene regulation will be for the study of and redundant gene and for the study of higher-order genetic and interactions. Mycobacterium tuberculosis and M. smegmatis at in or on with with and used at the and at sequences by all a and sequences the targets with the and target sequences the and the of the of by S. J. Kim J.S. A and that for of Cas9 RNA-guided PubMed Scopus Google Scholar), and with effects The by the of (11Zetsche B. Gootenberg J.S. Abudayyeh O.O. Slaymaker I.M. Makarova K.S. Essletzbichler P. Volz S.E. Joung J. van der Oost J. Regev A. Koonin E.V. Zhang F. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.Cell. 2015; 163: 759-771Abstract Full Text Full Text PDF PubMed Scopus (1983) Google Scholar), by the Hatfull with the and the E. coli of with the nuclease Asp917Ala and for expression in (17Choi S.Y. Woo H.M. CRISPRi-dCas12a: A dCas12a-mediated CRISPR interference for repression of multiple genes and metabolic engineering in Cyanobacteria.ACS Synth. Biol. 2020; 9: 2351-2361Crossref PubMed Scopus (16) Google Scholar) and into the expression of The by the in with the J.M. Hopkins F.F. Chavez A. Diallo M. Chase M.R. Gerrick E.R. Pritchard J.R. Church G.M. Rubin E.J. Sassetti C.M. Schnappinger D. Fortune S.M. Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform.Nat. Microbiol. 2017; 2: 16274Crossref PubMed Scopus (163) Google Scholar). The the control of the on an a The by the in C.J. Biswas S. Pakrasi H.B. Tunable repression of key photosynthetic processes using Cas12a CRISPR interference in the fast-growing Cyanobacterium Synechococcus sp. UTEX 2973.ACS Synth. Biol. 2020; 9: 132-143Crossref PubMed Scopus (14) Google Scholar) with the synthetic To crRNA into sequences by for the insertion site array and in a The using with and the and the The with and and a using to and and vectors used in this study are described in in for at least and to of and expression of the crRNA array and dCas12a with or with To into and using a For M. the at for the of the M. smegmatis to in with and to an of and for in the in and to an of with and to expression of and selection for the CRISPRi of into the and growth at by of in with or with and for to RNA in and using the and expression for each gene by using and to expression and as the of as in each strain relative to the with in and to RNA in with and using the The using the RNA and on the platform with The genomic and using the V. B. M. M. is for the to the mammalian 2015; PubMed Scopus Google Scholar, M. Chen J. R. J.C. H. W. T. M. transcriptional of a gene of 2011; PubMed Scopus Google Scholar), the using and using the analysis the using the cells into a and with for infected with at a of infection of The infection with of the or for by Mtb and by at and for This study and and are this This N. A. T. J. T. G.J. U. S. of for use with PubMed Scopus Google Scholar). The that they have of with the of this and Hatfull and for vectors and with and for with a a Hatfull G. N. F. N. F. and G. G. N. F. N. F. and G. G. G. N. F. G. N. F. and G. and This by and and by a by the to G. The is the of the and does not the of the of

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