Recent advances in genome-scale engineering in Escherichia coli and their applications

Abstract

Owing to the rapid advancement of genome engineering technologies, the scale of genome engineering has expanded dramatically. Genome editing has progressed from one genomic alteration at a time that could only be employed in few species, to the simultaneous generation of multiple modifications across many genomic loci in numerous species. The development and recent advances in multiplex automated genome engineering (MAGE)-associated technologies and clustered regularly interspaced short palindromic repeats and their associated protein (CRISPR-Cas)-based approaches, together with genome-scale synthesis technologies offer unprecedented opportunities for advancing genome-scale engineering in a broader range. These approaches provide new tools to generate strains with desired phenotypes, understand the complexity of biological systems, and directly evolve a genome with novel features. Here, we review the recent major advances in genome-scale engineering tools developed for Escherichia coli, focusing on their applications in identifying essential genes, genome reduction, recoding, and beyond.

Keywords

Genome-scale engineering / E. coli / Recombineering / CRISPR-Cas

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Given his role as editorial board member, Dr. Junbiao Dai, had no involvement in the peer-review of this article and has no access to information regarding its peer-review. Full responsibility for the editorial process for this article was delegated to Dr. Xiaoying Bian.

CRediT authorship contribution statement

Hui Gao: Conceptualization, Writing - original draft, Writing - review & editing, Funding acquisition. Zhichao Qiu: Writing - original draft, Writing - review & editing. Xuan Wang: Writing - original draft, Writing - review & editing. Xiyuan Zhang: Writing - review & editing. Yujia Zhang: Writing - original draft, Writing - review & editing. Junbiao Dai: Conceptualization, Writing - review & editing, Supervision, Funding acquisition. Zhuobin Liang: Conceptualization, Writing - review & editing, Supervision, Funding acquisition.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (2018YFA0903700), the National Natural Science Foundation of China (32030004, 32150025, 31901020), Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-PTJS-002), Guangdong Basic and Applied Basic Research Foundation (2023A1515030285), Shenzhen Science and Technology Program (KQTD20180413181837372) and Shenzhen Outstanding Talents Training Fund. Shenzhen Bay Laboratory startup funding.

References

[1] I. Thiele, B. Palsson, A protocol for generating a high-quality genome-scale metabolic reconstruction, Nat. Protoc. 5 (2010) 93-121, doi:10.1038/nprot.2009.203.

[2] J.D. Orth, T.M. Conrad, J. Na, J.A. Lerman, H. Nam, A.M. Feist, B. Palsson, A comprehensive genome-scale reconstruction of Escherichia coli metabolism-2011, Mol. Syst. Biol. 7 (2011) 1-9, doi:10.1038/msb.2011.65.

[3] S.K. Sharan, L.C. Thomason, S.G. Kuznetsov, D.L. Court, Recombineering: a homol- ogous recombination-based method of genetic engineering, Nat. Protoc. 4 (2009) 206-223, doi:10.1038/nprot.2008.227.

[4] H. Kurasawa, T. Ohno, R. Arai, Y. Aizawa, A guideline and challenges toward the minimization of bacterial and eukaryotic genomes, Curr. Opin. Syst. Biol. 24 (2020) 127-134, doi:10.1016/j.coisb.2020.10.012.

[5] S. Pontrelli, T.-Y. Chiu, E.I. Lan, F.Y.-H. Chen, P. Chang, J.C. Liao, Escherichia coli as a host for metabolic engineering, Metab. Eng. 50 (2018) 16-46, doi:10.1016/j.ymben.2018.04.008.

[6] Z.D. Blount, The natural history of model organisms: the unexhausted potential of E. coli, Elife. 4 (2015) e05826, doi:10.7554/eLife.05826.

[7] S. Huleani, M.R. Roberts, L. Beales, H. Emmanouil, Escherichia coli as an antibody expression host for the production of diagnostic proteins : significance and expression, Crit. Rev. Biotechnol. 42 (2022) 756-773, doi:10.1080/07388551.2021.1967871.

[8] T.S. Castiñeiras, S.G. Williams, A.G. Hitchcock, D.C. Smith, E. coli strain engineer- ing for the production of advanced biopharmaceutical products, FEMS Microbiol. Lett. 365 (2018) 1-10, doi:10.1093/femsle/fny162.

[9] M. Theisen, J.C. Liao, Industrial biotechnology: escherichia coli as a host, Ind. Biotechnol. (2017) 149-181, doi:10.1002/9783527807796.ch5.

[10] G.N.B. Chandresh Thakker, Irene Martínez, Ka-Yiu San, Succinate produc- tion in Escherichia coli, Biotechnol. J. 7 (2013) 213-224, doi:10.1002/biot. 201100061.

[11] D. McCloskey, B. Palsson, A.M. Feist, Basic and applied uses of genome-scale metabolic network reconstructions of Escherichia coli, Mol. Syst. Biol. 9 (2013) 1-15, doi:10.1038/msb.2013.18.

[12] L.C. Thomason, J.A. Sawitzke, X. Li, N. Costantino, D.L. Court, Recombineering: ge- netic engineering in bacteria using homologous recombination, Curr. Protoc. Mol. Biol. 106 (2014) 1.16. 1-1.16.39, doi:10.1002/0471142727.mb0116s106.

[13] D.L. Court, J.A. Sawitzke, L.C. Thomason, Genetic engineering using homolo- gous recombination, Annu. Rev. Genet. 36 (2002) 361-388, doi:10.1146/an- nurev.genet.36.061102.093104.

[14] Y. Daiguan, E.H. M, L. E-Chiang, J.N. A, C.N. G, C.D. L, An efficient recombination system for chromosome engineering in Escherichia coli, Proc. Natl. Acad. Sci. 97 (2000) 5978-5983, doi:10.1073/pnas.100127597.

[15] G. Pines, E.F. Freed, J.D. Winkler, R.T. Gill, Bacterial recombineering: genome en- gineering via phage-based homologous recombination, ACS Synth. Biol. 4 (2015) 1176-1185, doi:10.1021/acssynbio.5b00009.

[16] Z. Youming, B. Frank, M. Joep, S. Francis, A new logic for DNA engineering using recombination in Escherichia coli, Nat. Genet. 20 (1998) 123-128.

[17] R. Kolodner, S.D. Hall, C. Luisi-DeLuca, Homologous pairing proteins encoded by the Escherichia coli recE and recT genes, Mol. Microbiol. 11 (1994) 23-30, doi:10.1111/j.1365-2958.1994.tb00286.x.

[18] K.C. Murphy, Use of bacteriophage 𝜆recombination functions to promote gene re- placement in Escherichia coli, 180 (1998) 2063-2071.

[19] D. Yu, H.M. Ellis, E. Lee, N.A. Jenkins, N.G. Copeland, D.L. Court, An efficient recombination system for chromosome engineering in Escherichia coli, Proc. Natl. Acad. Sci. 97 (2000), doi:10.1073/pnas.100127597.

[20] M. Juhas, J.W. Ajioka, Lambda Red recombinase-mediated integration of the high molecular weight DNA into the Escherichia coli chromosome, Microb. Cell Fact. 15 (2016) 1-10, doi:10.1186/s12934-016-0571-y.

[21] D.M. Bubnov, T.V. Yuzbashev, T.V. Vybornaya, A.I. Netrusov, S.P. Sineoky, Development of new versatile plasmid-based systems for 𝜆 Red-mediated Es- cherichia coli genome engineering, J. Microbiol. Methods. 151 (2018) 48-56, doi:10.1016/j.mimet.2018.06.001.

[22] T.M. Wannier, P.N. Ciaccia, A.D. Ellington, G.T. Filsinger, F.J. Isaacs, K. Ja- vanmardi, M.A. Jones, A.M. Kunjapur, A. Nyerges, C. Pal, M.G. Schubert, G. M. Church, Recombineering and MAGE, Nat. Rev. Methods Prim. 1 (2021), doi:10.1038/s43586-020-00006-x.

[23] H.H. Wang, F.J. Isaacs, P.A. Carr, Z.Z. Sun, G. Xu, C.R. Forest, G.M. Church, Pro- gramming cells by multiplex genome engineering and accelerated evolution, Na- ture 460 (2009), doi:10.1038/nature08187.

[24] K.S. Makarova, Y.I. Wolf, J. Iranzo, S.A. Shmakov, O.S. Alkhnbashi, S.J.J. Brouns, E. Charpentier, D. Cheng, D.H. Haft, P. Horvath, S. Moineau, F.J.M. Mo- jica, D. Scott, S.A. Shah, V. Siksnys, M.P. Terns, Č. Venclovas, M.F. White, A.F. Yakunin, W. Yan, F. Zhang, R.A. Garrett, R. Backofen, J. van der Oost, R. Barrangou, E.V. Koonin, Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants, Nat. Rev. Microbiol. 18 (2020) 67-83, doi:10.1038/s41579-019-0299-x.

[25] A. Pickar-Oliver, C.A. Gersbach, The next generation of CRISPR-Cas tech- nologies and applications, Nat. Rev. Mol. Cell Biol. 20 (2019) 490-507, doi:10.1038/s41580-019-0131-5.

[26] A.C. Komor, A.H. Badran, D.R. Liu, CRISPR-based technologies for the manipulation of eukaryotic genomes, Cell 168 (2017) 20-36, doi:10.1016/j.cell.2016.10.044.

[27] J.D. Sander, J.K. Joung, CRISPR-Cas systems for editing, regulating and targeting genomes, Nat. Biotechnol. 32 (2014) 347-355, doi:10.1038/nbt.2842.

[28] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J.A. Doudna, E. Charpentier, A pro- grammable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity, Science (80-) 337 (2012) 816-822.

[29] X. Zhu, D. Zhao, H. Qiu, F. Fan, S. Man, C. Bi, The CRISPR /Cas9-facilitated multiplex pathway optimization (CFPO) technique and its application to improve the Escherichia coli xylose utilization pathway, Metab. Eng. 43 (2017) 37-45, doi:10.1016/j.ymben.2017.08.003.

[30] M.J. Lajoie, A.J. Rovner, D.B. Goodman, H. Aerni, A.D. Haimovich, G. Kuznetsov, J. a Mercer, H.H. Wang, P. a Carr, J. a Mosberg, N. Rohland, P.G. Schultz, J.M. Jacobson, J. Rinehart, G.M. Church, F.J. Isaacs, Genomically re- coded organisms expand biological functions, Science (80-) 342 (2013)357-360.

[31] N.J. Ma, D.W. Moonan, F.J. Isaacs, Precise manipulation of bacterial chromosomes by conjugative assembly genome engineering, Nat. Protoc. 9 (2014) 2285-2300, doi:10.1038/nprot.2014.081.

[32] M.G. Napolitano, M. Landon, C.J. Gregg, M.J. Lajoie, L. Govindarajan, J.A. Mos- berg, G. Kuznetsov, D.B. Goodman, O. Vargas-Rodriguez, F.J. Isaacs, D. Söll, G. M. Church, Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli, Proc. Natl. Acad. Sci. U. S. A. 113 (2016) E5588-E5597, doi:10.1073/pnas.1605856113.

[33] N.R. Sandoval, J.Y.H. Kim, T.Y. Glebes, P.J. Reeder, H.R. Aucoin, J.R. Warner, R.T. Gill, Strategy for directing combinatorial genome engineering in Es- cherichia coli, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 10540-10545, doi:10.1073/pnas.1206299109.

[34] S. Raman, J.K. Rogers, N.D. Taylor, G.M. Church, Evolution-guided optimization of biosynthetic pathways, Proc. Natl. Acad. Sci. U. S. A. 111 (2014) 17803-17808, doi:10.1073/pnas.1409523111.

[35] M. Amiram, A.D. Haimovich, C. Fan, Y.-S. Wang, H.-R. Aerni, I. Ntai, D.W. Moo- nan, N.J. Ma, A.J. Rovner, S.H. Hong, N.L. Kelleher, A.L. Goodman, M.C. Jewett, D. Söll, J. Rinehart, F.J. Isaacs, Evolution of translation machinery in recoded bac- teria enables multi-site incorporation of nonstandard amino acids, Nat. Biotechnol. 33 (2015) 1272-1279, doi:10.1038/nbt.3372.

[36] R.N.B. Isaacs FJ, P.A. Carr, H.H. Wang, M.J. Lajoie, B. Sterling, L. Kraal, A.C. Tolo- nen, T.A. Gianoulis, D.B. Goodman, Precise manipulation of chromosomes in vivo enables genome-wide codon replacement, Science (80-) 333 (2011) 348-353.

[37] C. Pa, Conditional DNA repair mutants enable highly precise genome engineering, Nucleic Acids Res 42 (2014), doi:10.1093/nar/gku105.

[38] R.M. Lennen, A.I.N. Wallin, M. Pedersen, M. Bonde, H. Luo, M.O.A. Sommer, M. J. Herrg, Transient overexpression of DNA adenine methylase enables efficient and mobile genome engineering with reduced off-target effects, Nucleic Acids Res 44 (2016) 1-14, doi:10.1093/nar/gkv1090.

[39] I. Nagy, B. Bálint, P. Bihari, V. Lázár, G. Apjok, A highly precise and portable genome engineering method allows comparison of mutational effects across bacte- rial species, Proc. Natl. Acad. Sci. 113 (2016), doi:10.1073/pnas.1520040113.

[40] A. Aronshtam, M.G. Marinus, Dominant negative mutator mutations in the mutL gene of Escherichia coli, Nucleic Acids Res. 24 (1996) 2498-2504.

[41] H.H. Wang, G. Xu, A.J. Vonner, G. Church, Modified bases enable high-efficiency oligonucleotide-mediated allelic replacement via mismatch repair evasion, Nucleic Acids Res. 39 (2011) 7336-7347, doi:10.1093/nar/gkr183.

[42] T.W. Van Ravesteyn, M. Dekker, A. Fish, T.K. Sixma, A. Wolters, R.J. Dekker, LNA modification of single-stranded DNA oligonucleotides allows subtle gene mod- ification in mismatch-repair-proficient cells, Proc. Natl. Acad. Sci 113 (2016), doi:10.1073/pnas.1513315113.

[43] J.A. Sawitzke, N. Costantino, X.T. Li, L.C. Thomason, M. Bubunenko, C. Court, D. L. Court, Probing cellular processes with oligo-mediated recombination and using the knowledge gained to optimize recombineering, J. Mol. Biol. 407(2011) 45-59, doi:10.1016/j.jmb.2011.01.030.

[44] T.M. Wannier, A. Nyerges, H.M. Kuchwara, M. Czikkely, D. Balogh, G.T. Filsinger, N. C. Borders, C.J. Gregg, M.J. Lajoie, X. Rios, C. Pál, G.M. Church, Improved bac- terial recombineering by parallelized protein discovery, Proc. Natl. Acad. Sci. U. S. A. 117 (2020) 13689-13698, doi:10.1073/pnas.2001588117.

[45] G.T. Filsinger, T.M. Wannier, F.B. Pedersen, I.D. Lutz, J. Zhang, D.A. Stork, A. Deb- nath, K. Gozzi, H. Kuchwara, V. Volf, S. Wang, X. Rios, C.J. Gregg, M.J. Lajoie, S. L. Shipman, J. Aach, M.T. Laub, G.M. Church, Characterizing the portability of phage-encoded homologous recombination proteins, Nat. Chem. Biol. 17 (2021) 394-402, doi:10.1038/s41589-020-00710-5.

[46] T.M. Wannier, A. Nyerges, H.M. Kuchwara, M. Czikkely, D. Balogh, Improved bac- terial recombineering by parallelized protein discovery, Proc. Natl. Acad. Sci. 117 (2020), doi:10.1073/pnas.2001588117.

[47] X. Zhu, Y. Wu, X. Lv, Y. Liu, G. Du, J. Li, L. Liu, Combining CRISPR-Cpf1 and recombineering facilitates fast and efficient genome editing in Escherichia coli, ACS Synth. Biol. (2022), doi:10.1021/acssynbio.2c00041.

[48] H.H. Wang, H. Kim, L. Cong, J. Jeong, D. Bang, G.M. Church, Genome-scale promoter engineering by coselection MAGE, Nat. Methods. 9 (2012) 591-593, doi:10.1038/nmeth.1971.

[49] P.A. Carr, H.H. Wang, B. Sterling, F.J. Isaacs, M.J. Lajoie, G. Xu, G.M. Church, J. M. Jacobson, Enhanced multiplex genome engineering through co- operative oligonucleotide co-selection, Nucleic Acids Res. 40 (2012), doi:10.1093/nar/gks455.

[50] C. Ronda, L.E. Pedersen, M.O.A. Sommer, A.T. Nielsen, CRMAGE: CRISPR opti- mized MAGE recombineering, Sci. Rep. 6 (2016) 1-11, doi:10.1038/srep19452.

[51] K. Umenhoffer, G. Draskovits, Á. Nyerges, I. Karcagi, B. Bogos, E. Tímár, B. Csörgö R. Herczeg, I. Nagy, T. Fehér, C. Pál, G. Pósfai, Genome-wide abolishment of mobile genetic elements using genome shuffling and CRISPR/Cas-assisted MAGE allows the efficient stabilization of a bacterial chassis, ACS Synth. Biol. 6 (2017) 1471-1483, doi:10.1021/acssynbio.6b00378.

[52] M. Quintin, N.J. Ma, S. Ahmed, S. Bhatia, A. Lewis, F.J. Isaacs, D. Densmore, Merlin: computer-aided oligonucleotide design for large scale genome engineering with mage, ACS Synth. Biol. 5 (2016) 452-458, doi:10.1021/acssynbio.5b00219.

[53] A.C. Komor, A.H. Badran, D.R. Liu, CRISPR-based technologies for the manipulation of eukaryotic genomes, Cell 168 (2017) 20-36, doi:10.1016/j.cell.2016.10.044.

[54] A. Pickar-Oliver, C.A. Gersbach, The next generation of CRISPR-Cas tech- nologies and applications, Nat. Rev. Mol. Cell Biol 20 (2019) 490-507, doi:10.1038/s41580-019-0131-5.

[55] C. Le, R.F. Ann, C. David, L. Shuailiang, B. Robert, H. Naomi, H.P. D, W. Xuebing, J. Wenyan, M.L. A, Z. Feng, Multiplex genome engineering using CRISPR/Cas sys- tems, Science (80-) 339 (2013) 819-823, doi:10.1126/science.1231143.

[56] W. Jiang, D. Bikard, D. Cox, F. Zhang, L.A. Marraffini, RNA-guided editing of bac- terial genomes using CRISPR-Cas systems, Nat. Biotechnol. 31 (2013) 233-239, doi:10.1038/nbt.2508.

[57] G. Liu, Q. Lin, S. Jin, C. Gao, The CRISPR-Cas toolbox and gene editing technologies, Mol. Cell. 82 (2022) 333-347, doi:10.1016/j.molcel.2021.12.002.

[58] Y. Jiang, B. Chen, C. Duan, B. Sun, J. Yang, S. Yang, Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system, Appl. Environ. Microbiol. 81(2015) 2506-2514, doi:10.1128/AEM.04023-14.

[59] S. Kiani, A. Chavez, M. Tuttle, R.N. Hall, R. Chari, D. Ter-ovanesyan, J. Qian, B.W. Pruitt, J. Beal, S. Vora, J. Buchthal, E.J.K. Kowal, M.R. Ebrahimkhani, J.J. Collins, R. Weiss, G. Church, Cas 9 gRNA engineering for genome editing, acti- vation and repression, Nat. Methods. 12 (2015), doi:10.1038/nmeth.3580.

[60] Y. Wang, Y. Wang, D. Pan, Y. Wang, Y. Wang, D. Pan, H. Yu, Y. Zhang, W. Chen, F. Li, Z. Wu, Guide RNA engineering enables efficient CRISPR editing with a minia- ture Syntrophomonas palmitatica Cas12f1 nuclease, CellReports 40(2022) 111418, doi:10.1016/j.celrep.2022.111418.

[61] J. Guo, T. Wang, C. Guan, B. Liu, C. Luo, Z. Xie, C. Zhang, X. Xing, Improved sgRNA design in bacteria via genome-wide activity profiling, Nucleic Acids Res 46 (2018) 7052-7069, doi:10.1093/nar/gky572.

[62] J. Wu, H. Yin, Engineering guide RNA to reduce the off-target effects of CRISPR, J. Genet. Genomics. 46(2019) 523-529, doi:10.1016/j.jgg.2019.11.003.

[63] D. Allen, M. Rosenberg, A. Hendel, Using synthetically engineered guide RNAs to enhance CRISPR genome editing systems in mammalian cells, Front, Genome Ed. 2 (2021) 1-16, doi:10.3389/fgeed.2020.617910.

[64] S. Bin Moon, D.Y. Kim, J.-H. Ko, J.-S. Kim, Y.-S. Kim, Improving CRISPR genome editing by engineering guide RNAs, Trends Biotechnol. 37 (2019) 870-881, doi:10.1016/j.tibtech.2019.01.009.

[65] T. Sakuma, A. Nishikawa, S. Kume, K. Chayama, T. Yamamoto, Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system, Sci. Rep. 4(2014) 4-9, doi:10.1038/srep05400.

[66] X. Feng, D. Zhao, X. Zhang, X. Ding, C. Bi, CRISPR/Cas 9 assisted multi- plex genome editing technique in Escherichia coli, Biotechnol. J. 13 (2018), doi:10.1002/biot.201700604.

[67] K. Xie, B. Minkenberg, Y. Yang, Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system, Proc. Natl. Acad. Sci. U. S. A. 112(2015) 3570-3575, doi:10.1073/pnas.1420294112.

[68] Q. Yuan, X. Gao, Multiplex base- and prime-editing with drive-and-process CRISPR arrays, Nat. Commun. 13 (2022) 1-13, doi:10.1038/s41467-022-30514-1.

[69] Y. Zhang, J. Wang, Z. Wang, Y. Zhang, S. Shi, J. Nielsen, Z. Liu, A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae, Nat. Commun. 10(2019) 1-10, doi:10.1038/s41467-019-09005-3.

[70] L. Song, J. Ouedraogo, M. Kolbusz, T. Truc, M. Nguyen, A. Tsang, Efficient genome editing using tRNA promoter-driven CRISPR /Cas9 gRNA in Aspergillus niger, PLoS ONE (2018) 1-17.

[71] C. Gene, G. Silencing, Development of a gRNA expression and processing platform for efficient CRISPR-Cas9-based gene editing and gene silencing in Candida tropi- calis, Microbiol. Spectr. 9 (2022) 1-14.

[72] A.D. Garst, M.C. Bassalo, G. Pines, S.A. Lynch, A.L. Halweg-Edwards, R. Liu, L. Liang, Z. Wang, R. Zeitoun, W.G. Alexander, R.T. Gill, Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engi- neering, Nat. Biotechnol. 35 (2017) 48-55, doi:10.1038/nbt.3718.

[73] D. Bikard, W. Jiang, P. Samai, A. Hochschild, F. Zhang, L.A. Marraffini, Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system, Nucleic Acids Res. 41 (2013) 7429-7437, doi:10.1093/nar/gkt520.

[74] R. Heler, P. Samai, J.W. Modell, C. Weiner, G.W. Goldberg, D. Bikard, L.A. Marraf- fini, Cas 9 specifies functional viral targets during CRISPR-Cas adaptation, Nature 519(2015) 199-202, doi:10.1038/nature14245.

[75] M.H. Larson, L.A. Gilbert, X. Wang, W.A. Lim, J.S. Weissman, L.S. Qi, CRISPR in- terference (CRISPRi) for sequence-specific control of gene expression, Nat. Protoc. 8 (2013) 2180-2196, doi:10.1038/nprot.2013.132.

[76] T. Wang, C. Guan, J. Guo, B. Liu, Y. Wu, Z. Xie, C. Zhang, X.H. Xing, Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance-net, Nat. Commun. 9 (2018), doi:10.1038/s41467-018-04899-x.

[77] L.S. Qi, M.H. Larson, L.A. Gilbert, J.A. Doudna, J.S. Weissman, A.P. Arkin, W.A. Lim, Repurposing CRISPR as an RNA-guided platform for sequence- specific control of gene expression, Cell 152 (2013) 1173-1183, doi:10.1016/j.cell.2013.02.022.

[78] J.M. Replogle, R.A. Saunders, A.N. Pogson, J.A. Hussmann, A. Lenail, A. Guna, L. Mascibroda, E.J. Wagner, K. Adelman, G. Lithwick-Yanai, N. Iremadze, F. Ober- strass, D. Lipson, J.L. Bonnar, M. Jost, T.M. Norman, J.S. Weissman, Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq, Cell (2022) 1-17, doi:10.1016/j.cell.2022.05.013.

[79] L. Fang, J. Fan, S. Luo, Y. Chen, C. Wang, Y. Cao, H. Song, Genome-scale target identification in Escherichia coli for high-titer production of free fatty acids, Nat. Commun. (2021), doi:10.1038/s41467-021-25243-w.

[80] A.C. Komor, Y.B. Kim, M.S. Packer, J.A. Zuris, D.R. Liu, Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage, Nature 533 (2016) 420-424, doi:10.1038/nature17946.

[81] N.M. Gaudelli, A.C. Komor, H.A. Rees, M.S. Packer, A.H. Badran, D.I. Bryson, D. R. Liu, Programmable base editing of A• T to G• C in genomic DNA without DNA cleavage, Nature 551 (2017) 464-471, doi:10.1038/nature24644.

[82] S. Banno, K. Nishida, T. Arazoe, H. Mitsunobu, A. Kondo, Deaminase-mediated multiplex genome editing in Escherichia coli, Nat. Microbiol. 3 (2018), doi:10.1038/s41564-017-0102-6.

[83] A.V. Anzalone, P.B. Randolph, J.R. Davis, A.A. Sousa, L.W. Koblan, J.M. Levy, P.J. Chen, C. Wilson, G.A. Newby, A. Raguram, D.R. Liu, Search-and-replace genome editing without double-strand breaks or donor DNA, Nature 576 (2019) 149-157, doi:10.1038/s41586-019-1711-4.

[84] Y. Tong, T.S. Jørgensen, C.M. Whitford, T. Weber, S.Y. Lee, A versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing, Nat. Commun. 12 (2021) 1-11, doi:10.1038/s41467-021-25541-3.

[85] E.V. Koonin, K.S. Makarova, E.V. Koonin, Origins and evolution of CRISPR-Cas systems, Philos. Trans. R. Soc. B. (2019).

[86] K. Chylinski, K.S. Makarova, E. Charpentier, E.V. Koonin, Classification and evo- lution of type II CRISPR-Cas systems, Nucleic Acids Res. 42 (2014) 6091-6105, doi:10.1093/nar/gku241.

[87] B. Zetsche, J.S. Gootenberg, O.O. Abudayyeh, I.M. Slaymaker, K.S. Makarova, P. Essletzbichler, S.E. Volz, J. Joung, J. Van Der Oost, A. Regev, E.V. Koonin, F. Zhang, Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system, Cell 163 (2015) 759-771, doi:10.1016/j.cell.2015.09.038.

[88] D.C. Swarts, M. Jinek, Mechanistic insights into the cis- and trans- acting DNase activities of Cas12a, Mol. Cell. 73(2019) 589-600 e4, doi:10.1016/j.molcel.2018.11.021.

[89] T. Karvelis, G. Bigelyte, J.K. Young, Z. Hou, R. Zedaveinyte, K. Budre, S. Paulraj, V. Djukanovic, S. Gasior, A. Silanskas, C. Venclovas, V. Siksnys, PAM recog- nition by miniature CRISPR-Cas12f nucleases triggers programmable double- stranded DNA target cleavage, Nucleic Acids Res. 48 (2020) 5016-5023, doi:10.1093/nar/gkaa208.

[90] W.X. Yan, P. Hunnewell, L.E. Alfonse, J.M. Carte, E. Keston-Smith, S. Sothisel- vam, A.J. Garrity, S. Chong, K.S. Makarova, E.V. Koonin, D.R. Cheng, D.A. Scott, Functionally diverse type V CRISPR-Cas systems, Science (80-) 363 (2019) 88-91, doi:10.1126/science.aav7271.

[91] W.X. Yan, S. Chong, H. Zhang, K.S. Makarova, E.V. Koonin, D.R. Cheng, D.A. Scott, Cas13d is a compact RNA-targeting type VI CRISPR effector positively modulated by a WYL-domain-containing accessory protein, Mol. Cell. 70(2018) 327-339 e5, doi:10.1016/j.molcel.2018.02.028.

[92] S. Kannan, H. Altae-Tran, X. Jin, V.J. Madigan, R. Oshiro, K.S. Makarova, E. V. Koonin, F. Zhang, Compact RNA editors with small Cas13 proteins, Nat. Biotechnol. 40(2022) 194-197, doi:10.1038/s41587-021-01030-2.

[93] O.O. Abudayyeh, J.S. Gootenberg, P. Essletzbichler, S. Han, J. Joung, J.J. Be- lanto, V. Verdine, D.B.T. Cox, M.J. Kellner, A. Regev, E.S. Lander, D.F. Voytas, A.Y. Ting, F. Zhang, RNA targeting with CRISPR-Cas13, Nature 550 (2017) 280-284, doi:10.1038/nature24049.

[94] O.O. Abudayyeh, J.S. Gootenberg, S. Konermann, J. Joung, I.M. Slaymaker, D. B.T. Cox, S. Shmakov, K.S. Makarova, E. Semenova, L. Minakhin, K. Severinov, A. Regev, E.S. Lander, E.V. Koonin, F. Zhang, C2c2 is a single-component pro- grammable RNA-guided RNA-targeting CRISPR effector, Science (80-) 353 (2016), doi:10.1126/science.aaf5573.

[95] K. Okano, Y. Sato, T. Hizume, K. Honda, Genome editing by miniature CRISPR/Cas12f1 enzyme in Escherichia coli, J. Biosci. Bioeng. 132(2021) 120-124, doi:10.1016/j.jbiosc.2021.04.009.

[96] X. Ao, Y. Yao, T. Li, T.T. Yang, X. Dong, Z.T. Zheng, G.Q. Chen, Q. Wu, Y. Guo, A multiplex genome editing method for Escherichia coli based on CRISPR-Cas12a, Front. Microbiol. 9 (2018) 1-13, doi:10.3389/fmicb.2018.02307.

[97] D.B.T. Cox, J.S. Gootenberg, O.O. Abudayyeh, B. Franklin, M.J. Kellner, J. Joung, F. Zhang, RNA editing with CRISPR-Cas13, Science (80-) 358 (2017) 1019-1027, doi:10.1126/science.aaq0180.

[98] B. Csörg ő, L.M. León, I.J. Chau-Ly, A. Vasquez-Rifo, J.D. Berry, C. Mahen- dra, E.D. Crawford, J.D. Lewis, J. Bondy-Denomy, A compact Cascade-Cas3 system for targeted genome engineering, Nat. Methods. 17(2020) 1183-1190, doi:10.1038/s41592-020-00980-w.

[99] J.E. Peters, K.S. Makarova, S. Shmakov, E.V. Koonin, Recruitment of CRISPR-Cas systems by Tn7-like transposons, Proc. Natl. Acad. Sci. U. S. A. 114 (2017) E7358-E7366, doi:10.1073/pnas.1709035114.

[100] S.E. Klompe, P.L.H. Vo, T.S. Halpin-Healy, S.H. Sternberg, Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration, Nature 571 (2019) 219-225, doi:10.1038/s41586-019-1323-z.

[101] P.L.H. Vo, C. Ronda, S.E. Klompe, E.E. Chen, C. Acree, H.H. Wang, S.H. Sternberg, CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering, Nat. Biotechnol. (2020), doi:10.1038/s41587-020-00745-y.

[102] A. Choudhury, J.A. Fenster, R.G. Fankhauser, J.L. Kaar, O. Tenaillon, R.T. Gill, CRISPR /Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli, Mol. Syst. Biol. 16(2020) 1-22, doi:10.15252/msb.20199265.

[103] W.E. Robertson, L.F.H. Funke, D. de la Torre, J. Fredens, K. Wang, J.W. Chin, Cre- ating custom synthetic genomes in Escherichia coli with REXER and GENESIS, Nat. Protoc. 16 (2021) 2345-2380, doi:10.1038/s41596-020-00464-3.

[104] J. Cello, A.V. Paul, E. Wimmer, Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template, Science (80-) 297 (2002) 1016-1018, doi:10.1126/science.1072266.

[105] D.G. Gibson, G.A. Benders, C. Andrews-Pfannkoch, E.A. Denisova, H. Baden-Tillson, J. Zaveri, T.B. Stockwell, A. Brownley, D.W. Thomas, M.A. Algire, C. Merryman, L. Young, V.N. Noskov, J.I. Glass, J.C. Venter, C.A. Hutchison, H.O. Smith, Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome, Science (80-) 319 (2008) 1215-1220, doi:10.1126/science. 1151721.

[106] D.G. Gibson, J.I. Glass, C. Lartigue, V.N. Noskov, R.Y. Chuang, M.A. Algire, G.A. Benders, M.G. Montague, L. Ma, M.M. Moodie, C. Merryman, S. Vashee, R. Kr- ishnakumar, N. Assad-Garcia, C. Andrews-Pfannkoch, E.A. Denisova, L. Young, Z.N. Qi, T.H. Segall-Shapiro, C.H. Calvey, P.P. Parmar, C.A. Hutchison, H.O. Smith, H. Gao, Z. Qiu, X. Wang, et al. J. C. Venter, Creation of a bacterial cell controlled by a chemically synthesized genome, Science (80-). 329 (2010) 52-56, doi:10.1126/science.1190719.

[107] C.A. Hutchison, R.Y. Chuang, V.N. Noskov, N. Assad-Garcia, T.J. Deerinck, M.H. El- lisman, J. Gill, K. Kannan, B.J. Karas, L. Ma, J.F. Pelletier, Z.Q. Qi, R.A. Richter, E.A. Strychalski, L. Sun, Y. Suzuki, B. Tsvetanova, K.S. Wise, H.O. Smith, J.I. Glass, C. Merryman, D.G. Gibson, J.C. Venter, Design and synthesis of a minimal bacterial genome, Science (80-) 351 (2016), doi:10.1126/science.aad6253.

[108] N. Annaluru, H. Muller, L.A. Mitchell, S. Ramalingam, G. Stracquadanio, S. M. Richardson, J.S. Dymond, Z. Kuang, L.Z. Scheifele, E.M. Cooper, Y. Cai, K. Zeller, N. Agmon, J.S. Han, M. Hadjithomas, J. Tullman, K. Caravelli, K. Cirelli, Z. Guo, V. London, A. Yeluru, S. Murugan, K. Kandavelou, N. Agier, G. Fis- cher, K. Yang, J.A. Martin, M. Bilgel, P. Bohutski, K.M. Boulier, B.J. Capaldo, J. Chang, K. Charoen, W.J. Choi, P. Deng, J.E. DiCarlo, J. Doong, J. Dunn, J.I. Fein- berg, C. Fernandez, C.E. Floria, D. Gladowski, P. Hadidi, I. Ishizuka, J. Jabbari, C.Y.L. Lau, P.A. Lee, S. Li, D. Lin, M.E. Linder, J. Ling, J. Liu, J. Liu, M. London, M. Henry, J. Mao, J.E. McDade, A. McMillan, A.M. Moore, W.C. Oh, Y. Ouyang, R. Patel, M. Paul, L.C. Paulsen, J. Qiu, A. Rhee, M.G. Rubashkin, I.Y. Soh, N.E. So- tuyo, V. Srinivas, A. Suarez, A. Wong, R. Wong, W.R. Xie, Y. Xu, A.T. Yu, R. Koszul, J.S. Bader, J.D. Boeke, S. Chandrasegaran, Total synthesis of a functional de- signer eukaryotic chromosome, Science (80-) 344 (2014) 55-58, doi:10.1126/sci- ence.1249252.

[109] W. Zhang, G. Zhao, Z. Luo, Y. Lin, L. Wang, Y. Guo, A. Wang, S. Jiang, Q. Jiang, J. Gong, Y. Wang, S. Hou, J. Huang, T. Li, Y. Qin, J. Dong, Q. Qin, J. Zhang, X. Zou, X. He, L. Zhao, Y. Xiao, M. Xu, E. Cheng, N. Huang, T. Zhou, Y. Shen, R. Walker, Y. Luo, Z. Kuang, L.A. Mitchell, K. Yang, S.M. Richardson, Y. Wu, B.-Z. Li, Y.-Y.- J. Yuan, H. Yang, J. Lin, G.-Q. Chen, Q. Wu, J.S. Bader, Y. Cai, J.D. Boeke, J. Dai, Engineering the ribosomal DNA in a megabase synthetic chromosome, Science (80-) 355 (2017) eaaf3981, doi:10.1126/science.aaf3981.

[110] Y. Shen, Y. Wang, T. Chen, F. Gao, J. Gong, D. Abramczyk, R. Walker, H. Zhao, S. Chen, W. Liu, Y. Luo, C.A. Müller, A. Paul-Dubois-Taine, B. Alver, G. Strac- quadanio, L.A. Mitchell, Z. Luo, Y. Fan, B. Zhou, B. Wen, F. Tan, Y. Wang, J. Zi, Z. Xie, B. Li, K. Yang, S.M. Richardson, H. Jiang, C.E. French, C.A. Nieduszyn- ski, R. Koszul, A.L. Marston, Y. Yuan, J. Wang, J.S. Bader, J. Dai, J.D. Boeke, X. Xu, Y. Cai, H. Yang, Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome, Science (80-) (2017) 355, doi:10.1126/science. aaf4791.

[111] L.A. Mitchell, A. Wang, G. Stracquadanio, Z. Kuang, X. Wang, K. Yang, S. Richard- son, J.A. Martin, Y. Zhao, R. Walker, Y. Luo, H. Dai, K. Dong, Z. Tang, Y. Yang, Y. Cai, A. Heguy, B. Ueberheide, D. Fenyö, J. Dai, J.S. Bader, J.D. Boeke, Synthesis, debugging, and effects of synthetic chromosome consolidation: synVI and beyond, Science (80-) 355 (2017), doi:10.1126/science.aaf4831.

[112] J. Dai, J.D. Boeke, Z. Luo, S. Jiang, Y. Cai, Sc3.0: revamping and minimizing the yeast genome, Genome Biol. 21 (2020) 1-4, doi:10.1186/s13059-020-02130-z.

[113] N. Ostrov, M. Landon, M. Guell, G. Kuznetsov, J. Teramoto, N. Cervantes, M. Zhou, K. Singh, M.G. Napolitano, M. Moosburner, E. Shrock, B.W. Pruitt, N. Conway, D.B. Goodman, C.L. Gardner, G. Tyree, A. Gonzales, B.L. Wanner, J.E. Norville, M.J. Lajoie, G.M. Church, Design, synthesis, and testing toward a 57-codon genome, Science (80-) 353 (2016) 819 LP -822, doi:10.1126/science.aaf3639.

[114] J. Fredens, K. Wang, D. de la Torre, L.F.H. Funke, W.E. Robertson, Y. Christova, T. Chia, W.H. Schmied, D.L. Dunkelmann, V. Beránek, C. Uttamapinant, A.G. Lla- mazares, T.S. Elliott, J.W. Chin, Total synthesis of Escherichia coli with a recoded genome, Nature 569 (2019) 514-518, doi:10.1038/s41586-019-1192-5.

[115] C.G. Acevedo-Rocha, G. Fang, M. Schmidt, D.W. Ussery, A. Danchin, From essential to persistent genes: a functional approach to constructing synthetic life, Trends Genet 29(2013) 273-279, doi:10.1016/j.tig.2012.11.001.

[116] Y.-C. Hwang, C.-C. Lin, J.-Y. Chang, H. Mori, H.-F. Juan, H.-C. Huang, Predicting essential genes based on network and sequence analysis, Mol. Biosyst. 5 (2009) 1672-1678, doi:10.1039/B900611G.

[117] L.A. Gallagher, J. Bailey, C. Manoil, Ranking essential bacterial processes by speed of mutant death, Proc. Natl. Acad. Sci. U. S. A. 117 (2020) 18010-18017, doi:10.1073/pnas.2001507117.

[118] J.M. Peters, A. Colavin, H. Shi, T.L. Czarny, M.H. Larson, S. Wong, J.S. Hawkins, C. H.S. Lu, B.M. Koo, E. Marta, A.L. Shiver, E.H. Whitehead, J.S. Weissman, E.D. Brown, L.S. Qi, K.C. Huang, C.A. Gross, A comprehensive, CRISPR-based functional analysis of essential genes in bacteria, Cell 165 (2016) 1493-1506, doi:10.1016/j.cell.2016.05.003.

[119] G. Giaever, A.M. Chu, L. Ni, C. Connelly, L. Riles, S. Véronneau, S. Dow, A. Lucau-Danila, K. Anderson, B. André, A.P. Arkin, A. Astromoff, M. El Bakkoury, R. Bang- ham, R. Benito, S. Brachat, S. Campanaro, M. Curtiss, K. Davis, A. Deutschbauer, K.- D. Entian, P. Flaherty, F. Foury, D.J. Garfinkel, M. Gerstein, D. Gotte, U. Güldener, J.H. Hegemann, S. Hempel, Z. Herman, D.F. Jaramillo, D.E. Kelly, S.L. Kelly, P. Köt- ter, D. LaBonte, D.C. Lamb, N. Lan, H. Liang, H. Liao, L. Liu, C. Luo, M. Lussier, R. Mao, P. Menard, S.L. Ooi, J.L. Revuelta, C.J. Roberts, M. Rose, P. Ross-Macdonald, B. Scherens, G. Schimmack, B. Shafer, D.D. Shoemaker, S. Sookhai-Mahadeo, R.K. Storms, J.N. Strathern, G. Valle, M. Voet, G. Volckaert, C. Wang, T.R. Ward, J. Wilhelmy, E.A. Winzeler, Y. Yang, G. Yen, E. Youngman, K. Yu, H. Bussey, J.D. Boeke, M. Snyder, P. Philippsen, R.W. Davis, M. Johnston, Func- tional profiling of the Saccharomyces cerevisiae genome, Nature 418 (2002) 387-391, doi:10.1038/nature00935.

[120] D.S. León, J. Nogales, Toward merging bottom -up and top -down model-based designing of synthetic microbial communities, Curr. Opin. Microbiol. 69(2022) 102169, doi:10.1016/j.mib.2022.102169.

[121] R. Maddamsetti, P.J. Hatcher, A.G. Green, B.L. Williams, D.S. Marks, R.E. Lenski, Core genes evolve rapidly in the long-term evolution experiment with Escherichia coli, Genome Biol. Evol. 9 (2017) 1072-1083, doi:10.1093/gbe/evx064.

[122] O. Lukjancenko, T.M. Wassenaar, D.W. Ussery, Comparison of 61 se- quenced Escherichia coli genomes, Microb. Ecol. 60 (2010) 708-720, doi:10.1007/s00248-010-9717-3.

[123] E. Martínez-Carranza, H. Barajas, L.D. Alcaraz, L. Servín-González, G.Y. Ponce-Soto, G. Soberón-Chávez, Variability of bacterial essential genes among closely related bacteria: the case of Escherichia coli, Front. Microbiol. 9 (2018) 1-7, doi:10.3389/fmicb.2018.01059.

[124] R.L. Charlebois, W.F. Doolittle, Computing prokaryotic gene ubiquity: rescuing the core from extinction, Genome Res. 14 (2004) 2469-2477, doi:10.1101/gr.3024704.

[125] C. Freiberg, B. Wieland, F. Spaltmann, K. Ehlert, H. Brötz, H. Labischinski, Iden- tification of novel essential Escherichia coli genes conserved among pathogenic bacteria, J. Mol. Microbiol. Biotechnol. 3 (2001) 483-489.

[126] E.V. Koonin, Comparative genomics, minimal gene-sets and the last universal com- mon ancestor, Nat. Rev. Microbiol. 1 (2003) 127-136, doi:10.1038/nrmicro751.

[127] T. Baba, T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K.A. Datsenko, M. Tomita, B.L. Wanner, H. Mori, Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection, Mol. Syst. Biol. 2 (2006), doi:10.1038/msb4100050.

[128] R. Zhang, Y. Lin, DEG 5.0, a database of essential genes in both prokaryotes and eukaryotes, Nucleic Acids Res. 37 (2009) 455-458, doi:10.1093/nar/gkn858.

[129] E.C.A. Goodall, A. Robinson, I.G. Johnston, S. Jabbari, K.A. Turner, A.F. Cunning- ham, P.A. Lund, J.A. Cole, I.R. Henderson, The essential genome of Escherichia coli K- 12, MBio 9 (2017) 1-18, doi:10.1101/237842.

[130] F. Rousset, L. Cui, E. Siouve, C. Becavin, F. Depardieu, D. Bikard, Genome-wide CRISPR-dCas9 screens in E. coli identify essential genes and phage host factors, PLoS Genet 14(2018) 1-28, doi:10.1371/journal.pgen.1007749.

[131] W. Tim, B. Kı vanç, H.N. W, K.K. M, P. Yorick, W.J. J, L.E. S, S.D. M, Identification and characterization of essential genes in the human genome, Science (80-) 350 (2015) 1096-1101, doi:10.1126/science.aac7041.

[132] K. Wang, J. Fredens, S.F. Brunner, S.H. Kim, T. Chia, J.W. Chin, Defining synony- mous codon compression schemes by genome recoding, Nature 539 (2016) 59-64, doi:10.1038/nature20124.

[133] J.F. Zürcher, W.E. Robertson, T. Kappes, G. Petris, T.S. Elliott, G.P.C. Salmond, J.W. Chin, Refactored genetic codes enable bidirectional genetic isolation, Science (80-). 378 (2022) 516-523, doi:10.1126/science.add8943.

[134] T. Mukai, T. Yoneji, K. Yamada, H. Fujita, S. Nara, M. Su’etsugu, Overcoming the challenges of megabase-sized plasmid construction in Escherichia coli, ACS Synth. Biol. 9 (2020) 1315-1327, doi:10.1021/acssynbio.0c00008.

[135] K. Wang, D. De Torre, W.E. Robertson, J.W. Chin, Programmed chromosome fission and fusion enable precise large-scale genome rearrangement and assembly, Science (80-) 926 (2019) 922-926.

[136] T. Yoneji, H. Fujita, T. Mukai, M. Su’etsugu, M. Su’etsugu, Grand scale genome manipulation via chromosome swapping in Escherichia coli programmed by three one megabase chromosomes, Nucleic Acids Res. 49 (2021) 1-12, doi:10.1093/nar/gkab298.

[137] R.T. Dame, F.Z.M. Rashid, D.C. Grainger, Chromosome organization in bacteria: mechanistic insights into genome structure and function, Nat. Rev. Genet. 21 (2020) 227-242, doi:10.1038/s41576-019-0185-4.

[138] V.S. Lioy, A. Cournac, M. Marbouty, S. Duigou, J. Mozziconacci, O. Espéli, F. Boccard, R. Koszul, Multiscale structuring of the E. coli chromosome by nucleoid-associated and condensin proteins, Cell 172(2018) 771-783 e18, doi:10.1016/j.cell.2017.12.027.

[139] R. Gil, F.J. Silva, J. Peretó, A. Moya, Determination of the core of a minimal bacterial gene set, Microbiol. Mol. Biol. Rev. 68 (2004) 518-537, doi:10.1128/mmbr.68.3.518-537.2004.

[140] L. Wang, C.D. Maranas, MinGenome: an in silico top-down approach for the syn- thesis of minimized genomes, ACS Synth. Biol. 7 (2018) 462-473, doi:10.1021/ac- ssynbio.7b00296.

[141] V. Kolisnychenko, G. Plunkett, C.D. Herring, T. Fehér, J. Pósfai, F.R. Blattner, G. Pósfai, Engineering a reduced Escherichia coli genome, Genome Res 12 (2002) 640-647, doi:10.1101/gr.217202.

[142] Y. Hirokawa, H. Kawano, K. Tanaka-Masuda, N. Nakamura, A. Nakagawa, M. Ito, H. Mori, T. Oshima, N. Ogasawara, Genetic manipulations restored the growth fitness of reduced-genome Escherichia coli, J. Biosci. Bioeng. 116 (2013) 52-58, doi:10.1016/j.jbiosc.2013.01.010.

[143] K. Standage-Beier, Q. Zhang, X. Wang, Targeted large-scale deletion of bac- terial genomes using CRISPR-nickases, ACS Synth. Biol. 4 (2015) 1217-1225, doi:10.1021/acssynbio.5b00132.

[144] Z. Zhang, A.E. Baxter, D. Ren, K. Qin, Z. Chen, S.M. Collins, H. Huang, C.A. Ko- mar, P.F. Bailer, J.B. Parker, G.A. Blobel, R.M. Kohli, E.J. Wherry, S.L. Berger, J. Shi, Efficient engineering of human and mouse primary cells using peptide-assisted genome editing, (2023). https://doi.org/10.1038/s41587-023-01756-1.