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Overview of guide RNA design tools for CRISPR-Cas9 genome editing technology

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Abstract

CRISPR-Cas (Clustered, Regularly Interspaced, Short Palindromic Repeats–CRISPR-associated (Cas)) RNA guided endonuclease has emerged as the most effective and widely used genome editing technology, which has become the most exciting and rapidly advancing research field. Efficient genome editing by the CRISPR-Cas9 system has been demonstrated in many species, and several laboratories have established CRISPR-Cas9 as a screening tool for systematic genetic analysis, similar to shRNA screening. At least three companies have been founded to leverage this technology for therapeutic uses. To facilitate the implementation of this technology, many software tools have been developed to identify guide RNAs that effectively target a desired genomic region. Here, I provide an overview of the technology, focusing on guide RNA design principles, available software tools and their strengths and weaknesses.

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References

  1. Bae S, Park J, Kim J S (2014). Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNAguided endonucleases. Bioinformatics, 30(10): 1473–1475

  2. Chen S, Sanjana N E, Zheng K, Shalem O, Lee K, Shi X, Scott D A, Song J, Pan J Q, Weissleder R, Lee H, Zhang F, Sharp P A (2015). Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell, 160(6): 1246–1260

  3. Cho S W, Kim S, Kim Y, Kweon J, Kim H S, Bae S, Kim J S (2014). Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res, 24(1): 132–141

  4. Chu S W, Noyes M B, Christensen R G, Pierce B G, Zhu L J, Weng Z, Stormo G D, Wolfe S A (2012). Exploring the DNA-recognition potential of homeodomains. Genome Res, 22(10): 1889–1898

  5. Cong L, Ran F A, Cox D, Lin S, Barretto R, Habib N, Hsu P D, Wu X, Jiang W, Marraffini L A, Zhang F (2013). Multiplex genome engineering using CRISPR/Cas systems. Science, 339(6121): 819–823

  6. Cradick T J, Qiu P, Lee CM, Fine E J, Bao G (2014). COSMID: AWebbased tool for identifying and validating CRISPR/Cas off-target sites. Mol Ther Nucleic Acids, 3(12): e214

  7. Ding Q, Regan S N, Xia Y, Oostrom L A, Cowan C A, Musunuru K (2013). Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell, 12(4): 393–394

  8. Doench J G, Hartenian E, Graham D B, Tothova Z, Hegde M, Smith I, Sullender M, Ebert B L, Xavier R J, Root D E (2014). Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol, 32(12): 1262–1267

  9. Doudna J A, Charpentier E (2014). Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213): 1258096

  10. Enuameh M S, Asriyan Y, Richards A, Christensen R G, Hall V L, Kazemian M, Zhu C, Pham H, Cheng Q, Blatti C, Brasefield J A, Basciotta M D, Ou J, McNulty J C, Zhu L J, Celniker S E, Sinha S, Stormo G D, Brodsky M H, Wolfe S A (2013). Global analysis of Drosophila Cys2-His2 zinc finger proteins reveals a multitude of novel recognition motifs and binding determinants. Genome Res, 23 (6): 928–940

  11. Esvelt K M, Mali P, Braff J L, Moosburner M, Yaung S J, Church G M (2013). Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods, 10(11): 1116–1121

  12. Friedland A E, Tzur Y B, Esvelt K M, Colaiácovo M P, Church G M, Calarco J A (2013). Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat Methods, 10(8): 741–743

  13. Fu Y, Sander J D, Reyon D, Cascio V M, Joung J K (2014). Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol, 32(3): 279–284

  14. Gratz S J, Cummings A M, Nguyen J N, Hamm D C, Donohue L K, Harrison M M, Wildonger J, O’Connor-Giles K M (2013). Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics, 194(4): 1029–1035

  15. Gupta A, Meng X, Zhu L J, Lawson N D, Wolfe S A (2011). Zinc finger protein-dependent and-independent contributions to the in vivo offtarget activity of zinc finger nucleases. Nucleic Acids Res, 39(1): 381–392

  16. Heigwer F, Kerr G, BoutrosM(2014). E-CRISP: fast CRISPR target site identification. Nat Methods, 11(2): 122–123

  17. Horvath P, Barrangou R (2010). CRISPR/Cas, the immune system of bacteria and archaea. Science, 327(5962): 167–170

  18. Hou Z, Zhang Y, Propson N E, Howden S E, Chu L F, Sontheimer E J, Thomson J A (2013). Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc Natl Acad Sci USA, 110(39): 15644–15649

  19. Hsu P D, Scott D A, Weinstein J A, Ran F A, Konermann S, Agarwala V, Li Y, Fine E J, Wu X, Shalem O, Cradick T J, Marraffini L A, Bao G, Zhang F (2013). DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol, 31(9): 827–832

  20. Hwang W Y, Fu Y, Reyon D, Maeder M L, Tsai S Q, Sander J D, Peterson R T, Yeh J R, Joung J K (2013). Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol, 31(3): 227–229

  21. Ikmi A, McKinney S A, Delventhal K M, Gibson M C (2014). TALEN and CRISPR/Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis. Nat Commun, 5: 5486

  22. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna J A, Charpentier E (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096): 816–821

  23. Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J (2013). RNAprogrammed genome editing in human cells. eLife, 2: e00471

  24. Joung J K, Sander J D (2013). TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol, 14(1): 49–55

  25. Koike-Yusa H, Li Y, Tan E P, Velasco-Herrera M C, Yusa K (2014). Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol, 32(3): 267–273

  26. Koonin E V, Makarova K S (2009). CRISPR-Cas: an adaptive immunity system in prokaryotes. F1000 Biol Rep, 1: 95

  27. Koonin E V, Makarova K S (2013). CRISPR-Cas: evolution of an RNAbased adaptive immunity system in prokaryotes. RNA Biol, 10(5): 679–686

  28. Li D, Qiu Z, Shao Y, Chen Y, Guan Y, Liu M, Li Y, Gao N, Wang L, Lu X, Zhao Y, Liu M (2013). Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat Biotechnol, 31(8): 681–683

  29. Lorenz R, Bernhart S H, Höner Zu, Siederdissen C, Tafer H, Flamm C, Stadler P F, Hofacker I L (2011). ViennaRNA Package 2.0. Algorithms Mol Biol, 6(1): 26

  30. Ma M, Ye A Y, Zheng W, Kong L (2013). A guide RNA sequence design platform for the CRISPR/Cas9 system for model organism genomes. BioMed Res Int, 2013: 270805

  31. Mali P, Aach J, Stranges P B, Esvelt K M, Moosburner M, Kosuri S, Yang L, Church G M (2013a). CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol, 31(9): 833–838

  32. Mali P, Yang L, Esvelt K M, Aach J, Guell M, DiCarlo J E, Norville J E, Church G M (2013b). RNA-guided human genome engineering via Cas9. Science, 339(6121): 823–826

  33. Meng X, Noyes MB, Zhu L J, Lawson N D, Wolfe S A (2008). Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat Biotechnol, 26(6): 695–701

  34. Prykhozhij S V, Rajan V, Gaston D, Berman J N (2015). CRISPR multitargeter: a web tool to find common and unique CRISPR single guide RNA targets in a set of similar sequences. PLoS ONE, 10(3): e0119372

  35. Ran F A, Hsu P D, Lin C Y, Gootenberg J S, Konermann S, Trevino A E, Scott D A, Inoue A, Matoba S, Zhang Y, Zhang F (2013a). Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell, 154(6): 1380–1389

  36. Ran F A, Hsu P D, Wright J, Agarwala V, Scott D A, Zhang F (2013b). Genome engineering using the CRISPR-Cas9 system. Nat Protoc, 8 (11): 2281–2308

  37. Sampson T R, Saroj S D, Llewellyn A C, Tzeng Y L, Weiss D S (2013). A CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature, 497(7448): 254–257

  38. Shalem O, Sanjana N E, Hartenian E, Shi X, Scott D A, Mikkelsen T S, Heckl D, Ebert B L, Root D E, Doench J G, Zhang F (2014). Genome-scale CRISPR-Cas9 knockout screening in human cells. Science, 343(6166): 84–87

  39. Smith C, Gore A, Yan W, Abalde-Atristain L, Li Z, He C, Wang Y, Brodsky R A, Zhang K, Cheng L, Ye Z (2014). Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs. Cell Stem Cell, 15 (1): 12–13

  40. Tsai S Q, Wyvekens N, Khayter C, Foden J A, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung J K (2014). Dimeric CRISPR RNAguided FokI nucleases for highly specific genome editing. Nat Biotechnol, 32(6): 569–576

  41. Tsai S Q, Zheng Z, Nguyen N T, Liebers M, Topkar V V, Thapar V, Wyvekens N, Khayter C, Iafrate A J, Le L P, Aryee M J, Joung J K (2015). GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol, 33(2): 187–197

  42. Wang T, Wei J J, Sabatini D M, Lander E S (2014). Genetic screens in human cells using the CRISPR-Cas9 system. Science, 343(6166): 80–84

  43. Wyman C, Kanaar R (2006). DNA double-strand break repair: all’s well that ends well. Annu Rev Genet, 40(1): 363–383

  44. Xiao A, Cheng Z, Kong L, Zhu Z, Lin S, Gao G, Zhang B (2014). CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics

  45. Xu H, Xiao T, Chen C H, Li W, Meyer C, Wu Q, Wu D, Cong L, Zhang F, Liu J S, Brown M, Liu S X (2015). Sequence determinants of improved CRISPR sgRNA design. Genome Res: gr.191452.115

  46. Yang H, Wang H, Shivalila C S, Cheng A W, Shi L, Jaenisch R (2013). One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell, 154(6): 1370–1379

  47. Zhu L J, Holmes B R, Aronin N, Brodsky M H (2014). CRISPRseek: a bioconductor package to identify target-specific guide RNAs for CRISPR-Cas9 genome-editing systems. PLoS ONE, 9(9): e108424

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Correspondence to Lihua Julie Zhu.

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Zhu, L.J. Overview of guide RNA design tools for CRISPR-Cas9 genome editing technology. Front. Biol. 10, 289–296 (2015). https://doi.org/10.1007/s11515-015-1366-y

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Keywords

  • CRISPR-Cas9
  • genome editing
  • gRNA design
  • off-target analysis
  • gRNA efficacy