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Methods of Evaluating the Efficiency of CRISPR/Cas Genome Editing

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Abstract—

The CRISPR/Cas system is currently widely used for genome editing. The procedure of genome editing includes two necessary steps: (i) searching for the most effective guide RNA, and (ii) analyzing clones for presence of the desired mutation. This review presents the methods used to assess the efficiency of the CRISPR/Cas system and to confirm mutation in the target locus and discusses their advantages and disadvantages. It aims to provide information that could help researchers to choose a technique most appropriate for their specific tasks and available resources.

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REFERENCES

  1. Gaj T., Sirk S.J., Shui S.L., Liu J. 2016. Genome-editing technologies: Principles and applications. Cold Spring Harb. Perspect. Biol.8 (12), pii: a023754. https://doi.org/10.1101/cshperspect.a023754

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Lomov N.A., Borunova V.V., Rubtsov M.A. 2015. CRISPR/Cas9 technology for targeted genome editing. Biopolym. Cell.31 (4), 243–248.

    Article  Google Scholar 

  3. Sorek R., Lawrence C.M., Wiedenheft B. 2013. CRISPR-mediated adaptive immune systems in Bacteria and Archaea.Annu. Rev. Biochem.82 (1), 237–266.

    Article  CAS  Google Scholar 

  4. Makarova K.S., Wolf Y.I., Koonin E.V. 2018. Classification and nomenclature of CRISPR-Cas systems: where from here? Cris. J.1 (5), 325–336.

    Article  Google Scholar 

  5. 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.

    Article  CAS  Google Scholar 

  6. Ran F.A., Hsu P.D., Wright J., Agarwala V., Scott D.A., Zhang F. 2013. Genome engineering using the CRIPR-Cas9 system. Nat. Protoc. 8 (11), 2281‒2308.

    Article  CAS  Google Scholar 

  7. Haeussler M., Schönig K., Eckert H., Eschstruth A., Mianné J., Renaud J.B., Schneider-Maunoury S., Shkumatava A., Teboul L., Kent J., Joly J.S., Concordet J.P. 2016. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol.17 (1), 148.

    Article  Google Scholar 

  8. Germini D., Tsfasman T., Zakharova V.V., Sjakste N., Lipinski M., Vassetzky Y. 2018. A comparison of techniques to evaluate the effectiveness of genome editing. Trends Biotechnol.36 (2), 147–159.

    Article  CAS  Google Scholar 

  9. Zischewski J., Fischer R., Bortesi L. 2017. Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases. Biotechnol. Adv.35 (1), 95–104.

    Article  CAS  Google Scholar 

  10. Hartlerode A., Scully R. 2009. Mechanisms of double-strand break repair in somatic mammalian cells. Biochem. J.423 (2), 157–168.

    Article  CAS  Google Scholar 

  11. Bell C.C., Magor G.W., Gillinder K.R., Perkins A.C. 2014. A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing. BMC Genomics. 15 (1), 1002.

    Article  Google Scholar 

  12. Sentmanat M.F., Peters S.T., Florian C.P., Connelly J.P., Pruett-Miller S.M. 2018. A survey of validation strategies for CRISPR-Cas9 editing. Sci. Rep.8 (1), 888.

    Article  Google Scholar 

  13. Vouillot L., Thélie A., Pollet N. 2015. Comparison of T7E1 and Surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3 (Bethesda). 5 (3), 407–415.

    Article  CAS  Google Scholar 

  14. Mashal R.D., Koontz J., Sklar J. 1995. Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases. Nat. Genet.9 (2), 177‒183.

    Article  CAS  Google Scholar 

  15. Qiu P., Shandilya H., D’Alessio J.M., O’Connor K., Durocher J., Gerard G.F. 2004. Mutation detection using SurveyorTM nuclease. BioTechniques. 36 (4), 702‒707.

    Article  CAS  Google Scholar 

  16. Saiki R.K., Scharf S., Faloona F., Mullis K.B., Horn G.T., Erlich H.A., Arnheim N. 1985. Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science.230 (4732), 1350‒1354.

    Article  CAS  Google Scholar 

  17. Urnov F.D., Miller J.C., Lee Y.L., Beausejour C.M., Rock J.M., Augustus S., Jamieson A.C., Porteus M.H., Gregory P.D., Holmes M.C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature. 435 (7042), 646‒651.

    Article  CAS  Google Scholar 

  18. Kim J.M., Kim D., Kim S., Kim J.S. 2014. Genotyping with CRISPR-Cas-derived RNA-guided endonucleases. Nat. Commun.5, 3157.

    Article  Google Scholar 

  19. Brinkman E.K., Chen T., Amendola M., van Steensel B. 2014. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res.42 (22), pii: e168. https://doi.org/10.1093/nar/gku936

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Yang Z., Steentoft C., Hauge C., Hansen L., Thomsen A.L., Niola F., Vester-Christensen M.B., Frödin M., Clausen H., Wandall H.H., Bennett E.P. 2015. Fast and sensitive detection of indels induced by precise gene targeting. Nucleic Acids Res.43 (9), pii: e59. https://doi.org/10.1093/nar/gkv126

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. VanLeuven A.J., Park S., Menke D.B, Lauderdale J.D. 2018. A PAGE screening approach for identifying CRISPR-Cas9-induced mutations in zebrafish. Biotechniques. 64 (6), 275–278.

    Article  CAS  Google Scholar 

  22. Pierce A.J., Johnson R.D., Thompson L.H., Jasin M. 1999. XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev.13 (20), 2633–2638.

    Article  CAS  Google Scholar 

  23. Menzorov A.G., Lukyanchikova V.A., Korablev A.N., Serova I.A., Fishman V.S. 2016. Genome editing using CRISPR/ Cas9 system: A practical guide. Russ. J. Genet.: Appl. Res.20 (6), 930–944.

    Google Scholar 

  24. Shahar O.D., Ram E.V.S.R., Shimshoni E., Hareli S., Meshorer E., Goldberg M. 2012. Live imaging of induced and controlled DNA double-strand break formation reveals extremely low repair by homologous recombination in human cells. Oncogene. 31 (30), 3495–3504.

    Article  CAS  Google Scholar 

  25. 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.

    Article  CAS  Google Scholar 

  26. Germini D., Bou Saada Y., Tsfasman T., Osina K., Robin C., Lomov N., Rubtsov M., Sjakste N., Lipinski M., Vassetzky Y. 2017. A one-step PCR-based assay to evaluate efficiency and precision of genomic DNA-editing tools. Mol. Ther. Methods Clin. Dev.5 (6), 43–50.

    Article  CAS  Google Scholar 

  27. Torres R., Martin M.C., Garcia A., Cigudosa J.C., Ramirez J.C., Rodriguez-Perales S. 2014. Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR-Cas9 system. Nat. Commun.5, 1–8.

    Article  Google Scholar 

  28. Pinheiro L.B., Coleman V.A., Hindson C.M., Herrmann J., Hindson B.J., Bhat S., Emslie K.R. 2012. Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal. Chem.84 (2), 1003–1011.

    Article  CAS  Google Scholar 

  29. Dabrowska M., Czubak K., Juzwa W., Krzyzosiak W.J., Olejniczak M., Kozlowski P. 2018. qEva-CRISPR: a method for quantitative evaluation of CRISPR/Cas-mediated genome editing in target and off-target sites. Nucleic Acids Res.46 (17), pii: e101. https://doi.org/10.1093/nar/gky505

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Schindelin J., Arganda-Carreras I., Frise E., Kaynig V., Longair M., Pietzsch T., Preibisch S., Rueden C., Saalfeld S., Schmid B., Tinevez J.Y., White D.J., Hartenstein V., Eliceiri K., Tomancak P., Cardona A. 2012. Fiji: an open-source platform for biological-image analysis. Nat. Methods. 9 (7), 676‒682.

    Article  CAS  Google Scholar 

  31. Harayama T., Riezman H. 2017. Detection of genome-edited mutant clones by a simple competition-based PCR method. PLoS One. 12 (6), 1–16.

    Article  Google Scholar 

  32. Yu C., Zhang Y., Yao S., Wei Y. 2014. A PCR based protocol for detecting indel mutations induced by TALENs and CRISPR/Cas9 in zebrafish. PLoS One. 9 (6), pii: e98282. https://doi.org/10.1371/journal.pone.0098282

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Thomas H.R., Percival S.M., Yoder B.K., Parant J.M. 2014. High-throughput genome editing and phenotyping facilitated by high resolution melting curve analysis. PLoS One. 9 (12), pii: e114632. https://doi.org/10.1371/journal.pone.0114632

    Article  Google Scholar 

  34. Dwight Z., Palais R., Wittwer C.T. 2011. uMELT: Prediction of high-resolution melting curves and dynamic melting profiles of PCR products in a rich web application. Bioinformatics. 27 (7), 1019‒1020.

    Article  CAS  Google Scholar 

  35. Dahlem T.J., Hoshijima K., Jurynec M.J., Gunther D., Starker C.G., Locke A.S., Weis A.M., Voytas D.F., Grunwald D.J. 2012. Simple methods for generating and detecting locus-specific mutations induced with TALENs in the Zebrafish genome. PLoS Genet.8 (8), pii: e1002861. https://doi.org/10.1371/journal.pgen.1002861

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Samarut E., Lissouba A., Drapeau P. 2016. A simplified method for identifying early CRISPR-induced indels in zebrafish embryos using High Resolution Melting analysis. BMC Genomics. 17, 547.

    Article  Google Scholar 

  37. Liew M., Pryor R., Palais R., Meadows C., Erali M., Lyon E., Wittwer C. 2004. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin. Chem.50 (7), 1156‒1164.

    Article  CAS  Google Scholar 

  38. Inazuka M., Wenz H.M., Sakabe M., Tahira T., Hayashi K. 1997. A streamlined mutation detection system: Multicolor post-PCR fluorescence labeling and single-strand conformational polymorphism analysis by capillary electrophoresis. Genome Res.7 (11), 1094–1103.

    Article  CAS  Google Scholar 

  39. Fennell J.P., Baker A.H., Dong Y., Zhu H. 2005. Single-strand conformational polymorphism analysis: Basic principles and routine practice. Hypertension. 108, 149‒158.

    Google Scholar 

  40. Zheng X., Yang S., Zhang D., Zhong Z., Tang X., Deng K., Zhou J., Qi Y., Zhang Y. 2016. Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism. Plant Cell Rep.35 (7), 1545–1554.

    Article  CAS  Google Scholar 

  41. Zhu X., Xu Y., Yu S., Lu L., Ding M., Cheng J., Song G., Gao X., Yao L., Fan D., Meng S., Zhang X., Hu S., Tian Y. 2014. An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system. Sci. Rep.4, 6420.

    Article  CAS  Google Scholar 

  42. Yu B., Sawyer N.A., Chiu C., Oefner P.J., Underhill P.A. 2006. DNA mutation detection using denaturing high-performance liquid chromatography (DHPLC). Curr. Protoc. Hum. Genet.7 (10), 1‒14.

    Google Scholar 

  43. Kc R., Srivastava A., Wilkowski J.M., Richter C.E., Shavit J.A., Burke D.T., Bielas S.L. 2016. Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection. Sci. Rep.6, 1–7.

    Article  Google Scholar 

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Funding

The work was with supported by the Russian Foundation for Basic Research, project nos. 19-04-00531_A and 19-54-16002_CNRS _A.

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Authors and Affiliations

  1. Moscow State University, 119991, Moscow, Russia

    N. A. Lomov, V. S. Viushkov, A. P. Petrenko, M. S. Syrkina & M. A. Rubtsov

  2. LIA LFR20 Laboratoire Franco-Russe de Recherches en Oncologie, Moscow State University, 119991, Moscow, Russia

    N. A. Lomov, V. S. Viushkov, M. S. Syrkina & M. A. Rubtsov

  3. Sechenov First Moscow State Medical University (Sechenov University), 119991, Moscow, Russia

    M. A. Rubtsov

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Correspondence to N. A. Lomov, V. S. Viushkov or M. A. Rubtsov.

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Translated by D. Timchenko

Abbreviations: DSB, double-strand break; PAGE, polyacrylamide gel electrophoresis; Cas9, CRISPR associated protein 9; CRISPR, clustered regularly interspaced short palindromic repeats; DHPLC, denaturing high-performance liquid chromatography; ENIT, engineered nuclease-induced translocations; FACS, fluorescence-activated cell sorting; gRNA, guide RNA; HRMA, high resolution melting analysis; IDAA, indel detection by amplicon analysis; LDR, ligation detection reaction; NHEJ, non-homologous end joining; RFLP, restriction fragment length polymorphism; SSCP, single-strand conformation polymorphism; TIDE, tracking of indels by decomposition.

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Lomov, N.A., Viushkov, V.S., Petrenko, A.P. et al. Methods of Evaluating the Efficiency of CRISPR/Cas Genome Editing. Mol Biol 53, 862–875 (2019). https://doi.org/10.1134/S0026893319060116

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