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This work used phage-encoded CRISPR/Cas to target plasmid-borne genes or chromosomal alleles conferring antibiotic resistance to E. coli cells. The phage-delivery of the specific RGENs was shown to improve host survival in an in vivo infection model as well.
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46•• Citorik, R.J., Mimee, M., Lu, T.K., Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat Biotechnol 32 (2014), 1141–1145 This work used phage-encoded CRISPR/Cas to target plasmid-borne genes or chromosomal alleles conferring antibiotic resistance to E. coli cells. The phage-delivery of the specific RGENs was shown to improve host survival in an in vivo infection model as well.
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This paper reports the elimination of antibiotic-resistant S. aureus cells from a mixed population by using phages to provide CRISPR/Cas cleavage of the resistance genes. The selective killing of resistant bacteria was also demonstrated in an in vivo mouse skin colonization model.
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50 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 81 (2015), 2506–2514.
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51 Wang, Y., Zhang, Z.-T., Seo, S.-O., Choi, K., Lu, T., Jin, Y.-S., Blaschek, H.P., Markerless chromosomal gene deletion in Clostridium beijerinckii using CRISPR/Cas9 system. J Biotechnol 200 (2015), 1–5.
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53 Tong, Y., Charusanti, P., Zhang, L., Weber, T., Lee, S.Y., CRISPR–Cas9 based engineering of actinomycetal genomes. ACS Synth Biol 4 (2015), 1020–1029.
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54 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., Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands. PLOS Genet, 9, 2013, e1003454.
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55
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This study targeted genomic islands of S. thermophilus using CRISPR/Cas to select for their spontaneous excision. Strains harboring deletions up to 102 kbp were generated this way, where IS elements flanking the islands had provided the homologies for recombination.
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55• Selle, K., Klaenhammer, T.R., Barrangou, R., CRISPR-based screening of genomic island excision events in bacteria. Proc Natl Acad Sci 112 (2015), 8076–8081 This study targeted genomic islands of S. thermophilus using CRISPR/Cas to select for their spontaneous excision. Strains harboring deletions up to 102 kbp were generated this way, where IS elements flanking the islands had provided the homologies for recombination.
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This work demonstrates that single-strand incisions of the E. coli chromosome, introduced by CRISPR/Cas nickases are not lethal, but in fact, can mediate homologous recombination between genomic repeats. Two deletions, totaling in length to 133 kbp were generated in one step using this technique.
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56• Standage-Beier, K., Zhang, Q., Wang, X., Targeted large-scale deletion of bacterial genomes using CRISPR-nickases. ACS Synth Biol 4 (2015), 1217–1225 This work demonstrates that single-strand incisions of the E. coli chromosome, introduced by CRISPR/Cas nickases are not lethal, but in fact, can mediate homologous recombination between genomic repeats. Two deletions, totaling in length to 133 kbp were generated in one step using this technique.
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59
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This study used CRISPR/Cas cleavage and dsDNA editing templates to generate barcoded deletions at three genomic loci of S. cerevisiae in parallel. In a directed in vivo protein evolution experiment, a mutant library of a cellobiose transporter gene was inserted into the chromosome and cells displaying improved cellobiose utilization were selected.
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59• Ryan, O.W., Skerker, J.M., Maurer, M.J., Li, X., Tsai, J.C., Poddar, S., Lee, M.E., DeLoache, W., Dueber, J.E., Arkin, A.P., et al. Selection of chromosomal DNA libraries using a multiplex CRISPR system. eLife, 3, 2014, e03703 This study used CRISPR/Cas cleavage and dsDNA editing templates to generate barcoded deletions at three genomic loci of S. cerevisiae in parallel. In a directed in vivo protein evolution experiment, a mutant library of a cellobiose transporter gene was inserted into the chromosome and cells displaying improved cellobiose utilization were selected.
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60 Bao, Z., Xiao, H., Liang, J., Zhang, L., Xiong, X., Sun, N., Si, T., Zhao, H., Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae. ACS Synth Biol 4 (2015), 585–594.
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61 Walter, J.M., Chandran, S.S., Horwitz, A.A., CRISPR-Cas-assisted multiplexing (CAM): simple same-day multi-locus engineering in yeast. J Cell Physiol, 2016, 10.1002/jcp.25375.
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62 Jakočiūnas, T., Rajkumar, A.S., Zhang, J., Arsovska, D., Rodriguez, A., Jendresen, C.B., Skjødt, M.L., Nielsen, A.T., Borodina, I., Jensen, M.K., et al. CasEMBLR: Cas9-facilitated multiloci genomic integration of in vivo assembled DNA parts in Saccharomyces cerevisiae. ACS Synth Biol 4 (2015), 1226–1234.
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63 Horwitz, A.A., Walter, J.M., Schubert, M.G., Kung, S.H., Hawkins, K., Platt, D.M., Hernday, A.D., Mahatdejkul-Meadows, T., Szeto, W., Chandran, S.S., et al. Efficient multiplexed integration of synergistic alleles and metabolic pathways in yeasts via CRISPR-Cas. Cell Syst 1 (2015), 88–96.
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