CRISPR-mediated host genomic DNA damage is efficiently repaired through microhomology-mediated end joining in Zymomonas mobilis

Xiaojie Wang; Bo Wu; Xin Sui; Zhufeng Zhang; Tao Liu; Yingjun Li; Guoquan Hu; Mingxiong He; Nan Peng

doi:10.1016/j.jgg.2021.02.012

Volume 48 Issue 2

Feb. 2021

Turn off MathJax

Article Contents

Article Navigation > Journal of Genetics and Genomics > 2021 > 48(2): 115-122

PDF( 1480 KB)

CRISPR-mediated host genomic DNA damage is efficiently repaired through microhomology-mediated end joining in Zymomonas mobilis

doi: 10.1016/j.jgg.2021.02.012

Xiaojie Wang^{a, #},
Bo Wu^{b, #},
Xin Sui^a,
Zhufeng Zhang^a,
Tao Liu^a,
Yingjun Li^a,
Guoquan Hu^b,
Mingxiong He^b,
Nan Peng^{a, b}

a
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China
b
Key Laboratory of Development and Application of Rural Renewable Energy (Ministry of Agriculture), Biomass Energy Technology Research Centre, Biogas Institute of Ministry of Agriculture, Chengdu, Sichuan 610041, China

More Information

Corresponding author: E-mail address: hemingxiong@caas.cn (Mingxiong He); E-mail address: nanp@mail.hzau.edu.cn (Nan Peng)
Received Date: 2020-11-01
Accepted Date: 2021-02-21
Rev Recd Date: 2021-02-07

Available Online: 2021-03-29

Publish Date: 2021-02-20

Abstract

Abstract

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against mobile genetic elements (MGEs) through uptake of invader-derived spacers. De novo adaptation samples spacers from both invaders and hosts, whereas primed adaptation shows higher specificity to sample spacers from invaders in many model systems as well as in the subtype I-F system of Zymomonas mobilis. Self-derived spacers will lead to CRISPR self-interference. However, our in vivo study demonstrated that this species used the microhomology-mediated end joining (MMEJ) pathway to efficiently repair subtype I-F CRISPR-Cas system-mediated DNA breaks guided by the self-targeting spacers. MMEJ repair of DNA breaks requires direct microhomologous sequences flanking the protospacers and leads to DNA deletions covering the protospacers. Importantly, CRISPR-mediated genomic DNA breaks failed to be repaired via MMEJ pathway in presence of higher copies of short homologous DNA. Moreover, CRISPR-cleaved exogenous plasmid DNA was failed to be repaired through MMEJ pathway, probably due to the inhibition of MMEJ by the presence of higher copies of the plasmid DNA inZ. mobilis. Our results infer that MMEJ pathway discriminates DNA damages between in the host chromosome versus mobile genetic element (MGE) DNA, and maintains genome stability post CRISPR immunity in Z. mobilis.
- CRISPR-Cas,
- CRISPR adaptation,
- Self-interference,
- Microhomology-mediated end joining

These authors contributed equally to this work.

FullText(HTML)

References(41)

References

[1]	Arslan, Z., Hermanns, V., Wurm, R., Wagner, R., Pul, U., 2014. Detection and characterization of spacer integration intermediates in type I-E CRISPR-Cas system. Nucleic Acids Res. 42, 7884-7893.
[2]	Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.A., Horvath, P., 2007. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709-1712.
[3]	Bernheim, A., Calvo-Villamanan, A., Basier, C., Cui, L., Rocha, E.P.C., Touchon, M., Bikard, D., 2017. Inhibition of NHEJ repair by type II-A CRISPR-Cas systems in bacteria. Nat. Commun. 8, 2094.
[4]	Brenac, L., Baidoo, E.E.K., Keasling, J.D., Budin, I., 2019. Distinct functional roles for hopanoid composition in the chemical tolerance of Zymomonas mobilis. Molecular microbiology 112, 1564-1575.
[5]	Brouns, S.J., Jore, M.M., Lundgren, M., Westra, E.R., Slijkhuis, R.J., Snijders, A.P., Dickman, M.J., Makarova, K.S., Koonin, E.V., van der Oost, J., 2008. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960-964.
[6]	Csorgo, B., Leon, L.M., Chau-Ly, I.J., Vasquez-Rifo, A., Berry, J.D., Mahendra, C., Crawford, E.D., Lewis, J.D., Bondy-Denomy, J., 2020. A compact Cascade-Cas3 system for targeted genome engineering. Nat. Methods 17, 1183-1190.
[7]	Datsenko, K.A., Pougach, K., Tikhonov, A., Wanner, B.L., Severinov, K., Semenova, E., 2012. Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nat. Commun. 3, 945.
[8]	Deveau, H., Barrangou, R., Garneau, J.E., Labonte, J., Fremaux, C., Boyaval, P., Romero, D.A., Horvath, P., Moineau, S., 2008. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. Journal of bacteriology 190, 1390-1400.
[9]	Dillard, K.E., Brown, M.W., Johnson, N.V., Xiao, Y., Dolan, A., Hernandez, E., Dahlhauser, S.D., Kim, Y., Myler, L.R., Anslyn, E.V., et al., 2018. Assembly and translocation of a CRISPR-Cas primed acquisition complex. Cell 175, 934-946.e15.
[10]	Fineran, P.C., Charpentier, E., 2012. Memory of viral infections by CRISPR-Cas adaptive immune systems: acquisition of new information. Virology 434, 202-209.
[11]	Fineran, P.C., Gerritzen, M.J., Suarez-Diez, M., Kunne, T., Boekhorst, J., van Hijum, S.A., Staals, R.H., Brouns, S.J., 2014. Degenerate target sites mediate rapid primed CRISPR adaptation. Proc. Natl. Acad. Sci. U. S. A. 111, E1629-1638.
[12]	Jackson, S.A., McKenzie, R.E., Fagerlund, R.D., Kieper, S.N., Fineran, P.C., Brouns, S.J., 2017. CRISPR-Cas: Adapting to change. Science 356, eaal5056.
[13]	Kerr, A.L., Jeon, Y.J., Svenson, C.J., Rogers, P.L., Neilan, B.A., 2011. DNA restriction-modification systems in the ethanologen, Zymomonas mobilis ZM4. Appl. Microbiol. Biotechnol. 89, 761-769.
[14]	Kunne, T., Kieper, S.N., Bannenberg, J.W., Vogel, A.I., Miellet, W.R., Klein, M., Depken, M., Suarez-Diez, M., Brouns, S.J., 2016. Cas3-derived target DNA degradation fragments fuel primed CRISPR adaptation. Mol. Cell 63, 852-864.
[15]	Lee, C., Kim, J., Shin, S.G., Hwang, S., 2006. Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli. J. Biotechnol. 123, 273-280.
[16]	Levy, A., Goren, M.G., Yosef, I., Auster, O., Manor, M., Amitai, G., Edgar, R., Qimron, U., Sorek, R., 2015. CRISPR adaptation biases explain preference for acquisition of foreign DNA. Nature 520, 505-510.
[17]	Li, M., Wang, R., Xiang, H., 2014a. Haloarcula hispanica CRISPR authenticates PAM of a target sequence to prime discriminative adaptation. Nucleic Acids Res. 42, 7226-7235.
[18]	Li, M., Wang, R., Zhao, D., Xiang, H., 2014b. Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process. Nucleic Acids Res. 42, 2483-2492.
[19]	Liu, T., Li, Y., Wang, X., Ye, Q., Li, H., Liang, Y., She, Q., Peng, N., 2015. Transcriptional regulator-mediated activation of adaptation genes triggers CRISPR de novo spacer acquisition. Nucleic Acids Res. 43, 1044-1055.
[20]	Liu, Z., Sun, M., Liu, J., Liu, T., Ye, Q., Li, Y., Peng, N., 2020. A CRISPR-associated factor Csa3a regulates DNA damage repair in Crenarchaeon Sulfolobus islandicus. Nucleic Acids Res. 48, 9681-9693.
[21]	Marraffini, L.A., Sontheimer, E.J., 2008. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322, 1843-1845.
[22]	Mojica, F.J., Diez-Villasenor, C., Garcia-Martinez, J., Almendros, C., 2009. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155, 733-740.
[23]	Pecoraro, V., Zerulla, K., Lange, C., Soppa, J., 2011. Quantification of ploidy in proteobacteria revealed the existence of monoploid, (mero-)oligoploid and polyploid species. PloS one 6, e16392.
[24]	Richter, C., Dy, R.L., McKenzie, R.E., Watson, B.N., Taylor, C., Chang, J.T., McNeil, M.B., Staals, R.H., Fineran, P.C., 2014. Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer. Nucleic Acids Res. 42, 8516-8526.
[25]	Savitskaya, E., Semenova, E., Dedkov, V., Metlitskaya, A., Severinov, K., 2013. High-throughput analysis of type I-E CRISPR/Cas spacer acquisition in E. coli. RNA Biol. 10, 716-725.
[26]	Semenova, E., Jore, M.M., Datsenko, K.A., Semenova, A., Westra, E.R., Wanner, B., van der Oost, J., Brouns, S.J., Severinov, K., 2011. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc. Natl. Acad. Sci. U. S. A. 108, 10098-10103.
[27]	Sfeir, A., Symington, L.S., 2015. Microhomology-Mediated End Joining: A Back-up Survival Mechanism or Dedicated Pathway? Trends Biochem. Sci. 40, 701-714.
[28]	Staals, R.H., Jackson, S.A., Biswas, A., Brouns, S.J., Brown, C.M., Fineran, P.C., 2016. Interference-driven spacer acquisition is dominant over naive and primed adaptation in a native CRISPR-Cas system. Nat. Commun. 7, 12853.
[29]	Stachler, A.E., Turgeman-Grott, I., Shtifman-Segal, E., Allers, T., Marchfelder, A., Gophna, U., 2017. High tolerance to self-targeting of the genome by the endogenous CRISPR-Cas system in an archaeon. Nucleic Acids Res. 45, 5208-5216.
[30]	Sun, B., Yang, J., Yang, S., Ye, R.D., Chen, D., Jiang, Y., 2018. A CRISPR-Cpf1-assisted non-homologous end joining genome editing system of Mycobacterium smegmatis. Biotechnology journal 13, e1700588.
[31]	Swarts, D.C., Mosterd, C., van Passel, M.W., Brouns, S.J., 2012. CRISPR interference directs strand specific spacer acquisition. PLoS One 7, e35888.
[32]	van der Oost, J., Jore, M.M., Westra, E.R., Lundgren, M., Brouns, S.J., 2009. CRISPR-based adaptive and heritable immunity in prokaryotes. Trends in biochemical sciences 34, 401-407.
[33]	Vercoe, R.B., Chang, J.T., Dy, R.L., Taylor, C., Gristwood, T., Clulow, J.S., Richter, C., Przybilski, R., Pitman, A.R., Fineran, P.C., 2013. Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands. PLoS Genet. 9, e1003454.
[34]	Watson, B.N.J., Easingwood, R.A., Tong, B., Wolf, M., Salmond, G.P.C., Staals, R.H.J., Bostina, M., Fineran, P.C., 2019. Different genetic and morphological outcomes for phages targeted by single or multiple CRISPR-Cas spacers. Philosophical transactions of the Royal Society of London. Series B, Biological sciences 374, 20180090.
[35]	Wei, Y., Terns, R.M., Terns, M.P., 2015. Cas9 function and host genome sampling in Type II-A CRISPR-Cas adaptation. Genes Dev. 29, 356-361.
[36]	Westra, E.R., Swarts, D.C., Staals, R.H., Jore, M.M., Brouns, S.J., van der Oost, J., 2012. The CRISPRs, they are a-changin': how prokaryotes generate adaptive immunity. Annu. Rev. Genet. 46, 311-339.
[37]	Yang, S., Mohagheghi, A., Franden, M.A., Chou, Y.C., Chen, X., Dowe, N., Himmel, M.E., Zhang, M., 2016. Metabolic engineering of Zymomonas mobilis for 2,3-butanediol production from lignocellulosic biomass sugars. Biotechnol. Biofuels 9, 189.
[38]	Yeh, C.D., Richardson, C.D., Corn, J.E., 2019. Advances in genome editing through control of DNA repair pathways. Nature Cell Biology 21, 1468-1478.
[39]	Yosef, I., Goren, M.G., Qimron, U., 2012. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res. 40, 5569-5576.
[40]	Zhang, Z., Pan, S., Liu, T., Li, Y., Peng, N., 2019. Cas4 Nucleases Can Effect Specific Integration of CRISPR Spacers. J. Bacteriol. 201, e00747-00718.
[41]	Zheng, Y., Han, J., Wang, B., Hu, X., Li, R., Shen, W., Ma, X., Ma, L., Yi, L., Yang, S., et al., 2019. Characterization and repurposing of the endogenous Type I-F CRISPR-Cas system of Zymomonas mobilis for genome engineering. Nucleic Acids Res. 47, 11461-11475.