HOW TO IMPROVE CRISPR SPECIFICITY - GENE EDITING EXPLAINED!
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- Опубліковано 11 лип 2024
- 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.
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.
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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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
doi.org/10.1016/j.cell.2016.1...
Music credits
“Go, Icarus! Go!” by The Whole Other
“New Year” by Bad Snacks
(UA-cam Audio Library) - Наука та технологія
Excellent video - thanks so much for posting this!
Glad you found it useful!
Many thanks! Will be usefull for review writing.
Wow nice video, thanks for helping me understand this topic
Glad to hear that
Excellent video - thanks very much for posting this! One question - what you are calling the 'protospacer' is really that portion of the 'non-target strand' of DNA opposite the 'target strand', the strand that the guide RNA 'spacer' binds to by complementary base-pairing. Protospacers are pieces of viral DNA that still have the PAM attached, and after the PAM is removed the 'spacers' are stored between the repeats, and a spacer and part of the repeat is transcribed into crRNA. So would it be more accurate to label your diagram 'non-target strand' rather than 'protospacer'? I know this is confusing, but apparently that is the terminology used these days?
“Protospacer’, ‘spacer’ and ‘target sequence’ are all terms that have been used generally when describing the first 20 bases of a sgRNA. I don’t disagree with you, but they all work if users know what those bases achieve.
@@GenomicsGurus Thanks for the quick reply!
Is the tail to tail guide orientation more efficient than prime editing?
We haven't compared the two. Primer editing should give much fewer unwanted edits, which is a big advantage in some applications