Frontiers in Biology

, Volume 10, Issue 4, pp 289–296 | Cite as

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

  • Lihua Julie ZhuEmail author


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.


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


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  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): 1473–1475PubMedCentralCrossRefPubMedGoogle Scholar
  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): 1246–1260CrossRefPubMedGoogle Scholar
  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): 132–141PubMedCentralCrossRefPubMedGoogle Scholar
  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): 1889–1898PubMedCentralCrossRefPubMedGoogle Scholar
  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): 819–823PubMedCentralCrossRefPubMedGoogle Scholar
  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): e214CrossRefGoogle Scholar
  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): 393–394PubMedCentralCrossRefPubMedGoogle Scholar
  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): 1262–1267PubMedCentralCrossRefPubMedGoogle Scholar
  9. Doudna J A, Charpentier E (2014). Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213): 1258096CrossRefPubMedGoogle Scholar
  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): 928–940PubMedCentralCrossRefPubMedGoogle Scholar
  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): 1116–1121CrossRefPubMedGoogle Scholar
  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): 741–743CrossRefPubMedGoogle Scholar
  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): 279–284PubMedCentralCrossRefPubMedGoogle Scholar
  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): 1029–1035PubMedCentralCrossRefPubMedGoogle Scholar
  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): 381–392PubMedCentralCrossRefPubMedGoogle Scholar
  16. Heigwer F, Kerr G, BoutrosM(2014). E-CRISP: fast CRISPR target site identification. Nat Methods, 11(2): 122–123CrossRefPubMedGoogle Scholar
  17. Horvath P, Barrangou R (2010). CRISPR/Cas, the immune system of bacteria and archaea. Science, 327(5962): 167–170CrossRefPubMedGoogle Scholar
  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): 15644–15649PubMedCentralCrossRefPubMedGoogle Scholar
  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): 827–832PubMedCentralCrossRefPubMedGoogle Scholar
  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): 227–229PubMedCentralCrossRefPubMedGoogle Scholar
  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: 5486CrossRefPubMedGoogle Scholar
  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): 816–821CrossRefPubMedGoogle Scholar
  23. Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J (2013). RNAprogrammed genome editing in human cells. eLife, 2: e00471PubMedCentralCrossRefPubMedGoogle Scholar
  24. Joung J K, Sander J D (2013). TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol, 14(1): 49–55PubMedCentralCrossRefPubMedGoogle Scholar
  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): 267–273CrossRefPubMedGoogle Scholar
  26. Koonin E V, Makarova K S (2009). CRISPR-Cas: an adaptive immunity system in prokaryotes. F1000 Biol Rep, 1: 95PubMedCentralPubMedGoogle Scholar
  27. Koonin E V, Makarova K S (2013). CRISPR-Cas: evolution of an RNAbased adaptive immunity system in prokaryotes. RNA Biol, 10(5): 679–686PubMedCentralCrossRefPubMedGoogle Scholar
  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): 681–683CrossRefPubMedGoogle Scholar
  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): 26PubMedCentralCrossRefPubMedGoogle Scholar
  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: 270805PubMedCentralPubMedGoogle Scholar
  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): 833–838CrossRefPubMedGoogle Scholar
  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): 823–826PubMedCentralCrossRefPubMedGoogle Scholar
  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): 695–701PubMedCentralCrossRefPubMedGoogle Scholar
  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): e0119372CrossRefGoogle Scholar
  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): 1380–1389PubMedCentralCrossRefPubMedGoogle Scholar
  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): 2281–2308PubMedCentralCrossRefPubMedGoogle Scholar
  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): 254–257PubMedCentralCrossRefPubMedGoogle Scholar
  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): 84–87PubMedCentralCrossRefPubMedGoogle Scholar
  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): 12–13PubMedCentralCrossRefPubMedGoogle Scholar
  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): 569–576PubMedCentralCrossRefPubMedGoogle Scholar
  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): 187–197PubMedCentralCrossRefPubMedGoogle Scholar
  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): 80–84PubMedCentralCrossRefPubMedGoogle Scholar
  43. Wyman C, Kanaar R (2006). DNA double-strand break repair: all’s well that ends well. Annu Rev Genet, 40(1): 363–383CrossRefPubMedGoogle Scholar
  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. BioinformaticsGoogle Scholar
  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.191452.115Google Scholar
  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): 1370–1379PubMedCentralCrossRefPubMedGoogle Scholar
  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): e108424CrossRefGoogle Scholar

Copyright information

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.Department of Molecular, Cell and Cancer Biology, Program in Bioinformatics and Integrated Biology, Program in Molecular MedicineUniversity of Massachusetts Medical SchoolWorcesterUSA

Personalised recommendations