Abstract
A critical stage in performing gene editing experiments using the CRISPR/Cas9 system is the design of guide RNA (gRNA). In this chapter, we conduct a review of the current gRNA design rules for maximizing on-target Cas9 activity while minimizing off-target activity. In addition, we present some of the currently available computational tools for gRNA activity prediction and assay design.
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Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213):1258096. https://doi.org/10.1126/science.1258096
Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315(5819):1709–1712. https://doi.org/10.1126/science.1138140
Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471(7340):602–607. https://doi.org/10.1038/nature09886
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821. https://doi.org/10.1126/science.1225829
Xu H, Xiao T, Chen CH, Li W, Meyer CA, Wu Q, Wu D, Cong L, Zhang F, Liu JS, Brown M, Liu XS (2015) Sequence determinants of improved CRISPR sgRNA design. Genome Res 25(8):1147–1157. https://doi.org/10.1101/gr.191452.115
Wong N, Liu W, Wang X (2015) WU-CRISPR: characteristics of functional guide RNAs for the CRISPR/Cas9 system. Genome Biol 16:218. https://doi.org/10.1186/s13059-015-0784-0
Wang T, Wei JJ, Sabatini DM, Lander ES (2014) Genetic screens in human cells using the CRISPR-Cas9 system. Science 343(6166):80–84. https://doi.org/10.1126/science.1246981
Moreno-Mateos MA, Vejnar CE, Beaudoin JD, Fernandez JP, Mis EK, Khokha MK, Giraldez AJ (2015) CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat Methods 12(10):982–988. https://doi.org/10.1038/nmeth.3543
Labuhn M, Adams FF, Ng M, Knoess S, Schambach A, Charpentier EM, Schwarzer A, Mateo JL, Klusmann JH, Heckl D (2018) Refined sgRNA efficacy prediction improves large- and small-scale CRISPR-Cas9 applications. Nucleic Acids Res 46(3):1375–1385. https://doi.org/10.1093/nar/gkx1268
Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, Sullender M, Ebert BL, Xavier RJ, Root DE (2014) Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol 32(12):1262–1267. https://doi.org/10.1038/nbt.3026
Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34(2):184–191. https://doi.org/10.1038/nbt.3437
Chuai G, Ma H, Yan J, Chen M, Hong N, Xue D, Zhou C, Zhu C, Chen K, Duan B, Gu F, Qu S, Huang D, Wei J, Liu Q (2018) DeepCRISPR: optimized CRISPR guide RNA design by deep learning. Genome Biol 19(1):80. https://doi.org/10.1186/s13059-018-1459-4
Chari R, Mali P, Moosburner M, Church GM (2015) Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach. Nat Methods 12(9):823–826. https://doi.org/10.1038/nmeth.3473
Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31(9):827–832. https://doi.org/10.1038/nbt.2647
Listgarten J, Weinstein M, Kleinstiver BP, Sousa AA, Joung JK, Crawford J, Gao K, Hoang L, Elibol M, Doench JG, Fusi N (2018) Prediction of off-target activities for the end-to-end design of CRISPR guide RNAs. Nat Biomed Eng 2(1):38–47. https://doi.org/10.1038/s41551-017-0178-6
Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA (2014) DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507(7490):62–67. https://doi.org/10.1038/nature13011
Anders C, Niewoehner O, Duerst A, Jinek M (2014) Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513(7519):569–573. https://doi.org/10.1038/nature13579
Sen D, Gilbert W (1988) Formation of parallel four-stranded complexes by guanine-rich motifs in DNA and its implications for meiosis. Nature 334(6180):364–366. https://doi.org/10.1038/334364a0
Nielsen S, Yuzenkova Y, Zenkin N (2013) Mechanism of eukaryotic RNA polymerase III transcription termination. Science 340(6140):1577–1580. https://doi.org/10.1126/science.1237934
Graf R, Li X, Chu VT, Rajewsky K (2019) sgRNA sequence motifs blocking efficient CRISPR/Cas9-mediated gene editing. Cell Rep 26(5):e1093–e1103. https://doi.org/10.1016/j.celrep.2019.01.024
Wu X, Scott DA, Kriz AJ, Chiu AC, Hsu PD, Dadon DB, Cheng AW, Trevino AE, Konermann S, Chen S, Jaenisch R, Zhang F, Sharp PA (2014) Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat Biotechnol 32(7):670–676. https://doi.org/10.1038/nbt.2889
Peng H, Zheng Y, Blumenstein M, Tao D, Li J (2018) CRISPR/Cas9 cleavage efficiency regression through boosting algorithms and Markov sequence profiling. Bioinformatics 34(18):3069–3077. https://doi.org/10.1093/bioinformatics/bty298
Ren X, Yang Z, Xu J, Sun J, Mao D, Hu Y, Yang SJ, Qiao HH, Wang X, Hu Q, Deng P, Liu LP, Ji JY, Li JB, Ni JQ (2014) Enhanced specificity and efficiency of the CRISPR/Cas9 system with optimized sgRNA parameters in Drosophila. Cell Rep 9(3):1151–1162. https://doi.org/10.1016/j.celrep.2014.09.044
Jiang F, Doudna JA (2017) CRISPR-Cas9 structures and mechanisms. Annu Rev Biophys 46:505–529. https://doi.org/10.1146/annurev-biophys-062215-010822
Zhao Y, Dai Z, Liang Y, Yin M, Ma K, He M, Ouyang H, Teng CB (2014) Sequence-specific inhibition of microRNA via CRISPR/CRISPRi system. Sci Rep 4:3943. https://doi.org/10.1038/srep03943
Jiang Q, Meng X, Meng L, Chang N, Xiong J, Cao H, Liang Z (2014) Small indels induced by CRISPR/Cas9 in the 5′ region of microRNA lead to its depletion and Drosha processing retardance. RNA Biol 11(10):1243–1249. https://doi.org/10.1080/15476286.2014.996067
Knight SC, Xie L, Deng W, Guglielmi B, Witkowsky LB, Bosanac L, Zhang ET, El Beheiry M, Masson JB, Dahan M, Liu Z, Doudna JA, Tjian R (2015) Dynamics of CRISPR-Cas9 genome interrogation in living cells. Science 350 (6262):823–826. https://doi.org/10.1126/science.aac6572
Horlbeck MA, Witkowsky LB, Guglielmi B, Replogle JM, Gilbert LA, Villalta JE, Torigoe SE, Tjian R, Weissman JS (2016) Nucleosomes impede Cas9 access to DNA in vivo and in vitro. elife 5. https://doi.org/10.7554/eLife.12677
Hartenian E, Doench JG (2015) Genetic screens and functional genomics using CRISPR/Cas9 technology. FEBS J 282(8):1383–1393. https://doi.org/10.1111/febs.13248
Haeussler M, Schonig K, Eckert H, Eschstruth A, Mianne J, Renaud JB, Schneider-Maunoury S, Shkumatava A, Teboul L, Kent J, Joly JS, Concordet JP (2016) Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol 17(1):148. https://doi.org/10.1186/s13059-016-1012-2
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Hiranniramol, K., Chen, Y., Wang, X. (2020). CRISPR/Cas9 Guide RNA Design Rules for Predicting Activity. In: Sioud, M. (eds) RNA Interference and CRISPR Technologies. Methods in Molecular Biology, vol 2115. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0290-4_19
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DOI: https://doi.org/10.1007/978-1-0716-0290-4_19
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