A CRISPR-nonhomologous end-joining-based strategy for rapid and efficient gene disruption in Mycobacterium abscessus

Abstract

Mycobacterium abscessus, a fast-growing, non-tuberculous mycobacterium resistant to most antimicrobial drugs, causes a wide range of serious infections in humans, posing a significant public health challenge. The development of effective genetic manipulation tools for M. abscessus is still in progress, limiting both research and therapeutic advancements. However, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) systems have emerged as promising tools for generating highly specific double-strand breaks (DSBs) in its genome. One of the mechanisms that repair these DSBs is the error-prone nonhomologous end-joining (NHEJ) pathway, which facilitates targeted gene editing. In this study, we introduced a novel application of the CRISPR-NHEJ approach in M. abscessus. We demonstrated that NrgA from M. marinum plays a crucial role in repairing DSBs induced by the CRISPR-Cas system in M. abscessus. Contrary to previous findings, our study also revealed that inhibiting or overexpressing components of homologous recombination/single-strand annealing significantly reduces the efficiency of NHEJ repair in M. abscessus. This discovery challenges current perspectives and suggests that NHEJ repair in M. abscessus may involve components from both homologous recombination and single-strand annealing pathways, highlighting the complex interactions among the three DSB repair mechanisms in M. abscessus.

Keywords

CRISPR-Cas12a / DSB repair / Mycobacterium abscessus / NHEJ / NrgA

References

[1] Victoria L, Gupta A, Gómez JL, Robledo J. Mycobacterium abscessus complex: a review of recent developments in an emerging pathogen. Front Cell Infect Microbiol. 2021; 11: 659997.

[2] Liu C-F, Song Y-M, He W-C, Liu D-X, He P, Bao J-J, et al. Nontuberculous mycobacteria in China: incidence and antimicrobial resistance spectrum from a nationwide survey. Infect Dis Poverty. 2021; 10: 59.

[3] Floto RA, Olivier KN, Saiman L, Daley CL, Herrmann J-L, Nick JA, et al. US Cystic Fibrosis Foundation and European Cystic Fibrosis Society consensus recommendations for the management of non-tuberculous mycobacteria in individuals with cystic fibrosis. Thorax. 2016; 71: 88-90.

[4] Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med. 2007; 175: 367-416.

[5] Nessar R, Cambau E, Reyrat JM, Murray A, Gicquel B. Mycobacterium abscessus: a new antibiotic nightmare. J Antimicrob Chemother. 2012; 67: 810-818.

[6] Mojica FJM, Montoliu L. On the origin of CRISPR-Cas technology: from prokaryotes to mammals. Trends Microbiol. 2016; 24: 811-820.

[7] Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015; 163: 759-771.

[8] Shmakov S, Smargon A, Scott D, Cox D, Pyzocha N, Yan W, et al. Diversity and evolution of class 2 CRISPR-Cas systems. Nat Rev Microbiol. 2017; 15: 169-182.

[9] Ceccaldi R, Rondinelli B, D'Andrea AD. Repair pathway choices and consequences at the double-strand break. Trends Cell Biol. 2016; 26: 52-64.

[10] Glickman MS. Double-strand DNA break repair in mycobacteria. Microbiol Spectr. 2014; 2: MGM2-0024-2013.

[11] Xue C, Greene EC. DNA repair pathway choices in CRISPR-Cas9-mediated genome editing. Trends Genet. 2021; 37: 639-656.

[12] Singh A. Guardians of the mycobacterial genome: a review on DNA repair systems in Mycobacterium tuberculosis. Microbiology. 2017; 163: 1740-1758.

[13] Gupta R, Barkan D, Redelman-Sidi G, Shuman S, Glickman MS. Mycobacteria exploit three genetically distinct DNA double-strand break repair pathways. Mol Microbiol. 2011; 79: 316-330.

[14] Brissett NC, Doherty AJ. Repairing DNA double-strand breaks by the prokaryotic non-homologous end-joining pathway. Biochem Soc Trans. 2009; 37: 539-545.

[15] Shuman S, Glickman MS. Bacterial DNA repair by non-homologous end joining. Nat Rev Microbiol. 2007; 5: 852-861.

[16] van Kessel JC, Hatfull GF. Recombineering in Mycobacterium tuberculosis. Nat Methods. 2007; 4: 147-152.

[17] Yan M-Y, Yan H-Q, Ren G-X, Zhao J-P, Guo X-P, Sun Y-C. CRISPR-Cas12a-assisted recombineering in bacteria. Appl Environ Microbiol. 2017; 83: e00947-17.

[18] Yan M-Y, Li S-S, Ding X-Y, Guo X-P, Jin Q, Sun Y-C. A CRISPR-Assisted nonhomologous end-joining strategy for efficient genome editing in Mycobacterium tuberculosis. mBio. 2020; 11: e02364-19.

[19] Wang S, Zhang J, Hameed HMA, Ding J, Guan P, Fang X, et al. Amino acid 17 in QRDR of gyrase A plays a key role in fluoroquinolones susceptibility in mycobacteria. Microbiol Spectr. 2023; 11: e0280923.

[20] Wang S, Cai X, Yu W, Zeng S, Zhang J, Guo L, et al. Arabinosyltransferase C mediates multiple drugs intrinsic resistance by altering cell envelope permeability in Mycobacterium abscessus. Microbiol Spectr. 2022; 10: e0276321.

[21] Chimukuche NM, Williams MJ. Genetic manipulation of non-tuberculosis mycobacteria. Front Microbiol. 2021; 12: 633510.

[22] Gao B, Ji R, Li Z, Su X, Li H, Sun Y, et al. Structural analysis and functional study of phosphofructokinase B (PfkB) from Mycobacterium marinum. Biochem Biophys Res Commun. 2021; 579: 129-135.

[23] Prasad D, Arora D, Nandicoori VK, Muniyappa K. Elucidating the functional role of Mycobacterium smegmatis recX in stress response. Sci Rep. 2019; 9: 10912.

[24] Prasad D, Muniyappa K. The extended N-terminus of Mycobacterium smegmatis RecX potentiates its ability to antagonize RecA functions. Biochim Biophys Acta Proteins Proteom. 2020; 1868: 140468.

[25] Tufariello JM, Malek AA, Vilchèze C, Cole LE, Ratner HK, González PA, et al. Enhanced specialized transduction using recombineering in Mycobacterium tuberculosis. mBio. 2014; 5: e01179-14-e79.

[26] Khanduja JS, Muniyappa K. Functional analysis of DNA replication fork reversal catalyzed by Mycobacterium tuberculosis RuvAB proteins. J Biol Chem. 2012; 287: 1345-1360.

[27] Reddy MS, Guhan N, Muniyappa K. Characterization of single-stranded DNA-binding proteins from mycobacteria. J Biol Chem. 2001; 276: 45959-45968.

[28] Ganesh N, Muniyappa K. Characterization of DNA strand transfer promoted by Mycobacterium smegmatis RecA reveals functional diversity with Mycobacterium tuberculosis RecA. Biochemistry. 2003; 42: 7216-7225.

[29] Kidane D, Sanchez H, Alonso JC, Graumann PL. Visualization of DNA double-strand break repair in live bacteria reveals dynamic recruitment of Bacillus subtilis RecF, RecO and RecN proteins to distinct sites on the nucleoids. Mol Microbiol. 2004; 52: 1627-1639.

[30] Gupta R, Ryzhikov M, Koroleva O, Unciuleac M, Shuman S, Korolev S, et al. A dual role for mycobacterial RecO in RecA-dependent homologous recombination and RecA-independent single-strand annealing. Nucleic Acids Res. 2013; 41: 2284-2295.

[31] Ryzhikov M, Gupta R, Glickman M, Korolev S. RecO protein initiates DNA recombination and strand annealing through two alternative DNA binding mechanisms. J Biol Chem. 2014; 289: 28846-28855.

[32] Griffith DE. Mycobacterium abscessus and antibiotic resistance: same as it ever was. Clin Infect Dis. 2019; 69: 1687-1689.

[33] Medjahed H, Singh AK. Genetic manipulation of Mycobacterium abscessus. Curr Protoc Microbiol. 2010; Chapter 10: Unit 10D.2.

[34] Viljoen A, Gutiérrez AV, Dupont C, Ghigo E, Kremer L. A simple and rapid gene disruption strategy in Mycobacterium abscessus: on the design and application of glycopeptidolipid mutants. Front Cell Infect Microbiol. 2018; 8: 69.

[35] Liu C, Yue Y, Xue Y, Zhou C, Ma Y. CRISPR-Cas9 assisted non-homologous end joining genome editing system of Halomonas bluephagenesis for large DNA fragment deletion. Microb Cell Fact. 2023; 22: 211.

[36] Cui L, Bikard D. Consequences of Cas9 cleavage in the chromosome of Escherichia coli. Nucleic Acids Res. 2016; 44: 4243-4251.

[37] Aniukwu J, Glickman MS, Shuman S. The pathways and outcomes of mycobacterial NHEJ depend on the structure of the broken DNA ends. Genes Dev. 2008; 22: 512-527.

[38] Guo J, Wang C, Han Y, Liu Z, Wu T, Liu Y, et al. Identification of lysine acetylation in Mycobacterium abscessus using LC-MS/MS after immunoprecipitation. J Proteome Res. 2016; 15: 2567-2578.

[39] Chhotaray C, Wang S, Tan Y, Ali A, Shehroz M, Fang C, et al. Comparative analysis of whole-genome and methylome profiles of a smooth and a rough Mycobacterium abscessus clinical strain. G3. 2020; 10: 13-22.

[40] Goude R, Roberts DM, Parish T. Electroporation of mycobacteria. Methods Mol Biol. 2015; 1285: 117-130.

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