HOW TO IMPROVE CRISPR SPECIFICITY - GENE EDITING EXPLAINED!

This series of short presentations on gene editing is brought to you by Dr Adam West, College of Medical Veterinary and Life Sciences at the University of Glasgow, Scotland. https://www.gla.ac.uk/people/adamwest This presentation describes four strategies that can be used to greatly improve the specificity of the S.pyogenes CRISPR/Cas9 system during genome editing. This include the use of the following 8:00 Dual nickases 12:55 Making a gene knock out cell lines with dual nickases 20:23 Enhanced specificity Cas9 mutants This is part of a series (   • Genome Editing Explained  ) that also covers Zinc finger nucleases (ZFNs) TALE nucleases (TALENs) CRISPR adaptive immune systems Using CRISPR/Cas9 for genome editing Precision editing Homology directed repair Prime editing This presentation is primarily aimed at university students, researchers, clinicians and journalists interested in fields related to molecular biology and genetics. It is for education purposes only. Please leave a comment to let us know whether this was helpful to you and what you think we should cover next. We are a new channel so please give us a like and share our video on your social media if you think others should see it. We have a lot of content coming up, so please subscribe! More accessible presentations for a wide audience can be found in the “Explained Simply” section of this channel in the near future. Artwork is © Adam West with the exception of images and data taken from publications referenced in these slides. References to reviews and journal articles are denoted by circled numbers at the bottom right of the slides. Links to these numbered articles are listed below. We recommend these as your best way to begin further reading on this subject. Publication List 21. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014 Jun 5;157(6):1262-78. https://doi.org/10.1016/j.cell.2014.0... 22. Hendel A, Fine EJ, Bao G, Porteus MH. Quantifying on- and off-target genome editing. Trends Biotechnol. 2015 Feb;33(2):132-40. https://doi.org/10.1016/j.tibtech.201... 23. Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M, Carte J, Chen W, Roark N, Ranganathan S, Ravinder N, Chesnut JD. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol. 2015 Aug 20;208:44-53. https://doi.org/10.1016/j.jbiotec.201... 24. Brinkman EK, Chen T, Amendola M, van Steensel B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 2014 Dec 16;42(22):e168. https://doi.org/10.1093/nar/gku936 25. Koo T, Lee J, Kim JS. Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9. Mol Cells. 2015 Jun;38(6):475-81. https://doi.org/10.14348/molcells.201... 30. Howden SE, McColl B, Glaser A, Vadolas J, Petrou S, Little MH, Elefanty AG, Stanley EG. A Cas9 Variant for Efficient Generation of Indel-Free Knockin or Gene-Corrected Human Pluripotent Stem Cells. Stem Cell Reports. 2016 Sep 13;7(3):508-517. https://doi.org/10.1016/j.stemcr.2016... 31. Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol. 2014 Mar;32(3):279-284. https://doi.org/10.1038/nbt.2808 32. Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. Rationally engineered Cas9 nucleases with improved specificity. Science. 2016 Jan 1;351(6268):84-8. https://doi.org/10.1126/science.aad5227 33. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, Joung JK. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 2016 Jan 28;529(7587):490-5. https://doi.org/10.1038/nature16526 34. Komor AC, Badran AH, Liu DR. CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes. Cell. 2017 Jan 12;168(1-2):20-36. https://doi.org/10.1016/j.cell.2016.1... Music credits “Go, Icarus! Go!” by The Whole Other “New Year” by Bad Snacks (YouTube Audio Library)