2022, Volume 15, Issue 8
Engineering >> 2022, Volume 15, Issue 8 doi: 10.1016/j.eng.2022.02.013
PAM-Expanded Streptococcus thermophilus Cas9 C-to-T and C-to-G Base Editors for Programmable Base Editing in Mycobacteria
a School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China
b University of Chinese Academy of Sciences, Beijing 100049, China
c Clinical Research Center, the Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing 210003, China
d Department of Tuberculosis, the Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing 210003, China
e Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
f Guangzhou Laboratory, Guangzhou 510120, China
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Abstract
New therapeutic strategies for the rapid and effective treatment of drug-resistant tuberculosis are highly desirable, and their development can be drastically accelerated by facile genetic manipulation methods in Mycobacterium tuberculosis (M. tuberculosis). Clustered regularly interspaced short palindromic repeat (CRISPR) base editors allow for rapid, robust, and programmed single-base substitutions and gene inactivation, yet no such systems are currently available in M. tuberculosis. By screening distinct CRISPR base editors, we discovered that only the unusual Streptococcus thermophilus CRISPR associated protein 9 (St1Cas9) cytosine base editor (CBE)—but not the widely used Streptococcus pyogenes Cas9 (SpCas9) or Lachnospiraceae bacterium Cpf1 (LbCpf1) CBEs—is active in mycobacteria. Despite the notable C-to-T conversions, a high proportion of undesired byproducts exists with St1Cas9 CBE. We therefore engineered St1Cas9 CBE by means of uracil DNA glycosylase inhibitor (UGI) or uracil DNA glycosylase (UNG) fusion, yielding two new base editors (CTBE and CGBE) capable of C-to-T or C-to-G conversions with dramatically enhanced editing product purity and multiplexed editing capacity in Mycobacterium smegmatis (M. smegmatis). Because wild-type St1Cas9 recognizes a relatively strict protospacer adjacent motif (PAM) sequence for DNA targeting, we engineered a PAM-expanded St1Cas9 variant by means of structureguided protein engineering for the base editors, substantially broadening the targeting scope. We first developed and characterized CTBE and CGBE in M. smegmatis, and then applied CTBE for genome editing in M. tuberculosis. Our approaches significantly reduce the efforts and time needed for precise genetic manipulation and will facilitate functional genomics, antibiotic-resistant mechanism study, and drugtarget exploration in M. tuberculosis and related organisms.
Keywords
CRISPR ; Cas9 ; Mycobacterium tuberculosis ; Genome editing ; Base editing
SupplementaryMaterials
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References
[ 1 ] World Health Organization. Global tuberculosis report 2018. Report. 2018.
[ 2 ] Gandhi NR, Nunn P, Dheda K, Schaaf HS, Zignol M, van Soolingen D, et al. Multidrug-resistant and extensively drug-resistant tuberculosis: a threat to global control of tuberculosis. Lancet 2010;375(9728):1830–43. link1
[ 3 ] Udwadia ZF, Amale RA, Ajbani KK, Rodrigues C. Totally drug-resistant tuberculosis in India. Clin Infect Dis 2012;54(4):579–81. link1
[ 4 ] Balasubramanian V, Pavelka MS, Bardarov SS, Martin J, Weisbrod TR, McAdam RA, et al. Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates. J Bacteriol 1996;178(1):273–9. link1
[ 5 ] Bardarov S, Bardarov S, Pavelka MS, Sambandamurthy V, Larsen M, Tufariello J, et al. Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis. Microbiology 2002;148(Pt 10):3007–17. link1
[ 6 ] van Kessel JC, Hatfull GF. Recombineering in Mycobacterium tuberculosis. Nat Methods 2007;4(2):147–52. link1
[ 7 ] Murphy KC, Nelson SJ, Nambi S, Papavinasasundaram K, Baer CE, Sassetti CM. ORBIT: a new paradigm for genetic engineering of mycobacterial chromosomes. MBio 2018;9(6):e01467-18. link1
[ 8 ] Yan M, Li S, Ding X, Guo X, Jin Q, Sun Y. A CRISPR-assisted nonhomologous endjoining strategy for efficient genome editing in Mycobacterium tuberculosis. MBio 2020;11(1):e02364-19. link1
[ 9 ] Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012;337(6096):816–21. link1
[10] Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013;339(6121):819–23. link1
[11] Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science 2013;339(6121):823–6. link1
[12] Ge X, Xi H, Yang F, Zhi X, Fu Y, Chen D, et al. CRISPR/Cas9-AAV mediated knockin at NRL locus in human embryonic stem cells. Mol Ther Nucleic Acids 2016;5:e393. link1
[13] Cobb RE, Wang Y, Zhao H. High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system. ACS Synth Biol 2015;4(6):723–8. link1
[14] Yang F, Liu C, Chen D, Tu M, Xie H, Sun H, et al. CRISPR/Cas9-loxP-mediated gene editing as a novel site-specific genetic manipulation tool. Mol Ther Nucleic Acids 2017;7:378–86. link1
[15] Tong Y, Charusanti P, Zhang L, Weber T, Lee SY. CRISPR-Cas9 based engineering of actinomycetal genomes. ACS Synth Biol 2015;4(9):1020–9. link1
[16] Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 2013;31 (3):233–9. link1
[17] Chen W, Zhang Y, Yeo WS, Bae T, Ji Q. Rapid and efficient genome editing in Staphylococcus aureus by using an engineered CRISPR/Cas9 system. J Am Chem Soc 2017;139(10):3790–5. link1
[18] Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013;152(5):1173–83. link1
[19] Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, et al. CRISPRmediated modular RNA-guided regulation of transcription in eukaryotes. Cell 2013;154(2):442–51. link1
[20] Choudhary E, Thakur P, Pareek M, Agarwal N. Gene silencing by CRISPR interference in mycobacteria. Nat Commun 2015;6(1):6267. link1
[21] Singh AK, Carette X, Potluri LP, Sharp JD, Xu R, Prisic S, et al. Investigating essential gene function in Mycobacterium tuberculosis using an efficient CRISPR interference system. Nucleic Acids Res 2016;44(18):e143. link1
[22] Rock JM, Hopkins FF, Chavez A, Diallo M, Chase MR, Gerrick ER, et al. Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform. Nat Microbiol 2017;2(4):16274. link1
[23] Fleck N, Grundner C. A Cas12a-based CRISPR interference system for multigene regulation in mycobacteria. J Biol Chem 2021;297(2):100990. link1
[24] Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 2016;533(7603):420–4. link1
[25] Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, et al. Programmable base editing of A●T to G●C in genomic DNA without DNA cleavage. Nature 2017;551(7681):464–71. link1
[26] Jin S, Zong Y, Gao Q, Zhu Z, Wang Y, Qin P, et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 2019;364 (6437):292–5. link1
[27] Tong Y, Whitford CM, Robertsen HL, Blin K, Jørgensen TS, Klitgaard AK, et al. Highly efficient DSB-free base editing for Streptomycetes with CRISPR-BEST. Proc Natl Acad Sci USA 2019;116(41):20366–75. link1
[28] Gu T, Zhao S, Pi Y, Chen W, Chen C, Liu Q, et al. Highly efficient base editing in Staphylococcus aureus using an engineered CRISPR RNA-guided cytidine deaminase. Chem Sci 2018;9(12):3248–53. link1
[29] Wang Y, Wang Z, Chen Y, Hua X, Yu Y, Ji Q. A highly efficient CRISPR-Cas9- based genome engineering platform in Acinetobacter baumannii to understand the H2O2-sensing mechanism of OxyR. Cell Chem Biol 2019;26(12):1732–42. link1
[30] Zheng K, Wang Y, Li N, Jiang F, Wu C, Liu F, et al. Highly efficient base editing in bacteria using a Cas9-cytidine deaminase fusion. Commun Biol 2018;1:32. link1
[31] Li Q, Seys FM, Minton NP, Yang J, Jiang Y, Jiang W, et al. CRISPR-Cas9D10A nickase-assisted base editing in the solvent producer Clostridium beijerinckii. Biotechnol Bioeng 2019;116(6):1475–83. link1
[32] Chen W, Zhang Y, Zhang Y, Pi Y, Gu T, Song L, et al. CRISPR/Cas9-based genome editing in Pseudomonas aeruginosa and cytidine deaminase-mediated base editing in Pseudomonas species. iScience 2018;6:222–31. link1
[33] Wang Y, Wang S, Chen W, Song L, Zhang Y, Shen Z, et al. CRISPR-Cas9 and CRISPR-assisted cytidine deaminase enable precise and efficient genome editing in Klebsiella pneumoniae. Appl Environ Microbiol 2018;84(23): e01834–18. link1
[34] Banno S, Nishida K, Arazoe T, Mitsunobu H, Kondo A. Deaminase-mediated multiplex genome editing in Escherichia coli. Nat Microbiol 2018;3(4):423–9. link1
[35] Wang Y, Liu Y, Liu J, Guo Y, Fan L, Ni X, et al. MACBETH: multiplex automated Corynebacterium glutamicum base editing method. Metab Eng 2018;47:200–10. link1
[36] Gibson DG, Young L, Chuang RY, Venter JC, Hutchison 3rd CA, Smith HO. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 2009;6(5):343–5. link1
[37] Kluesner MG, Nedveck DA, Lahr WS, Garbe JR, Abrahante JE, Webber BR, et al. EditR: a method to quantify base editing from sanger sequencing. CRISPR J 2018;1(3):239–50. link1
[38] Altenbuchner J. Editing of the bacillus subtilis genome by the CRISPR-Cas9 system. Appl Environ Microbiol 2016;82(17):5421–7. link1
[39] Huang H, Chai C, Li N, Rowe P, Minton NP, Yang S, et al. CRISPR/Cas9-based efficient genome editing in Clostridium ljungdahlii, an autotrophic gasfermenting bacterium. ACS Synth Biol 2016;5(12):1355–61. link1
[40] Leenay RT, Vento JM, Shah M, Martino ME, Leulier F, Beisel CL. Genome editing with CRISPR-Cas9 in Lactobacillus plantarum revealed that editing outcomes can vary across strains and between methods. Biotechnol J 2019;14 (3):1700583. link1
[41] Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 2015;81(7):2506–14. link1
[42] Li X, Wang Y, Liu Y, Yang B, Wang X, Wei J, et al. Base editing with a Cpf1- cytidine deaminase fusion. Nat Biotechnol 2018;36(4):324–7. link1
[43] Zhang Y, Zhang H, Xu X, Wang Y, Chen W, Wang Y, et al. Catalytic-state structure and engineering of Streptococcus thermophilus Cas9. Nat Catal 2020;3 (10):813–23. link1
[44] Komor AC, Zhao KT, Packer MS, Gaudelli NM, Waterbury AL, Koblan LW, et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T: a base editors with higher efficiency and product purity. Sci Adv 2017;3(8):eaao4774. link1
[45] Zhao D, Li J, Li S, Xin X, Hu M, Price MA, et al. Glycosylase base editors enable Cto-A and C-to-G base changes. Nat Biotechnol 2021;39(1):35–40. link1
[46] Kurt IC, Zhou R, Iyer S, Garcia SP, Miller BR, Langner LM, et al. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat Biotechnol 2021;39(1):41–6. link1
[47] Chen L, Park JE, Paa P, Rajakumar PD, Prekop HT, Chew YT, et al. Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins. Nat Commun 2021;12(1):1384. link1
[48] Koblan LW, Arbab M, Shen MW, Hussmann JA, Anzalone AV, Doman JL, et al. Efficient C●G-to-G●C base editors developed using CRISPRi screens, targetlibrary analysis, and machine learning. Nat Biotechnol 2021;39(11):1414–25. link1
[49] Billon P, Bryant EE, Joseph SA, Nambiar TS, Hayward SB, Rothstein R, et al. CRISPR-mediated base editing enables efficient disruption of eukaryotic genes through induction of STOP codons. Mol Cell 2017;67(6):1068–79. link1
[50] Yu H, Wu Z, Chen X, Ji Q, Tao S. CRISPR-CBEI: a designing and analyzing tool kit for cytosine base editor-mediated gene inactivation. mSystems 2020;5(5): e00350-20. link1
[51] Gupta HK, Shrivastava S, Sharma R. A novel calcium uptake transporter of uncharacterized P-type ATPase family supplies calcium for cell surface integrity in Mycobacterium smegmatis. MBio 2017;8(5):e01388-17. link1
[52] Unissa AN, Subbian S, Hanna LE, Selvakumar N. Overview on mechanisms of isoniazid action and resistance in Mycobacterium tuberculosis. Infect Genet Evol 2016;45:474–92. link1
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