Abstract
RNA-guided Cas9 nucleases derived from clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems have recently been adapted as sequence-programmable tools for various purposes such as genome editing and transcriptional regulation. A critical aspect of the system is the selection and validation of spacer sequences that allow precise targeting of the guide RNA-Cas9 complex. We describe a procedure involving computational and experimental steps to identify and test potentially interesting spacer sequences in bacterial genomes.
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References
Esvelt KM, Wang HH (2013) Genome-scale engineering for systems and synthetic biology. Mol Syst Biol 9:641. doi:10.1038/msb.2012.66
Marraffini L, Sontheimer EJ (2010) CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet 11:181–190. doi:10.1038/nrg2749
Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327:167–170. doi:10.1126/science.1179555
Jiang W, Bikard D, Cox D et al (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31:233–239. doi:10.1038/nbt.2508
Mali P, Esvelt KM, Church GM (2013) Cas9 as a versatile tool for engineering biology. Nat Methods 10:957–963. doi:10.1038/nmeth.2649
Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32:347–355. doi:10.1038/nbt.2842
Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278. doi:10.1016/j.cell.2014.05.010
Gasiunas G, Barrangou R, Horvath P, Siksnys V (2012) PNAS Plus: Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci 109:E2579–E2586. doi:10.1073/pnas.1208507109
Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–822
Esvelt KM, Mali P, Braff JL et al (2013) Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods 10:1116–1121. doi:10.1038/nmeth.2681
Anders C, Niewoehner O, Duerst A, Jinek M (2014) Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513:569–573. doi:10.1038/nature13579
Nishimasu H, Ran FA, Hsu PD et al (2014) Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156:935–949. doi:10.1016/j.cell.2014.02.001
Mali P, Aach J, Stranges PB et al (2013) Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31:833–838. doi:10.1038/nbt.2675
Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823. doi:10.1126/science.1231143
Qi LS, Larson MH, Gilbert L et al (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152:1173–1183. doi:10.1016/j.cell.2013.02.022
Bikard D, Jiang W, Samai P et al (2013) Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res 41:7429–7437. doi:10.1093/nar/gkt520
Tanenbaum ME, Gilbert L, Qi LS et al (2014) A versatile protein tagging system for signal amplification in single molecule imaging and gene regulation. Cell 159:635–646. doi:10.1016/j.cell.2014.09.039
Grenier F, Matteau D, Baby V, Rodrigue S (2014) Complete genome sequence of Escherichia coli BW25113. Genome Announc 2:e01038. doi:10.1128/genomeA.01038-14
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645. doi:10.1073/pnas.120163297
Datta S, Costantino N, Court DL (2006) A set of recombineering plasmids for gram-negative bacteria. Gene 379:109–115. doi:10.1016/j.gene.2006.04.018
Schleif R (2010) AraC protein, regulation of the l-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action. FEMS Microbiol Rev 34:779–796. doi:10.1111/j.1574-6976.2010.00226.x
Mathews DH, Sabina J, Zuker M et al (1999) Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol 288:911–940
Acknowledgement
We are grateful to Donald L. Court (NCI-Frederick) for the generous gift of pSIM7 and to Dominick Matteau and Alain Lavigueur for critical reading of the manuscript. We thank the Centre de calcul scientifique of Université de Sherbrooke for computational resources and technical support. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). S.R. holds a Chercheur-boursier Junior 1 award from the Fonds de recherche Québec-Santé.
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Grenier, F., Lucier, JF., Rodrigue, S. (2015). Selection and Validation of Spacer Sequences for CRISPR-Cas9 Genome Editing and Transcription Regulation in Bacteria. In: Leblanc, B., Rodrigue, S. (eds) DNA-Protein Interactions. Methods in Molecular Biology, vol 1334. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2877-4_15
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DOI: https://doi.org/10.1007/978-1-4939-2877-4_15
Publisher Name: Humana Press, New York, NY
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