Designing and Executing Prime Editing Experiments in Mammalian Cells

Join us to discuss cutting-edge technologies and advances in the biomedical and life sciences!
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🧬 Prime editing (PE) is a precision gene editing technology that enables the programmable installation of substitutions, insertions and deletions without requiring double-strand DNA breaks (DSBs). Prime editing has already been used in many basic science applications and has also been used to rescue cell and animal models of genetic disease. Furthermore, enhanced prime editing systems drastically improve editing efficiencies relative to the originally reported prime editor, and new applications such as twin prime editing (twinPE) can precisely insert or delete hundreds of base pairs of DNA.
Both PE and twinPE can also be used in tandem with recombinases to achieve gene-sized (over 5 kb) insertions and inversions. Achieving optimal PE requires careful experimental design, and the large number of parameters that influence PE outcomes can be daunting. In this webinar, we will discuss current best practices for designing and carrying out PE and twinPE experiments.
Stay connected with our lab for future prime editing updates!
💙 Twitter ➡ / davidrliu AND / liugroup
🧬 Lab website ➡ www.liugroup.us/
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⏰ Timestamps
0:00 Introduction
3:28 Overview of genome editing methods
6:41 Overview of prime editing (PE) method
12:57 Step 1: Select a target protospacer
14:16 Step 2: Design RTT and PBS
18:47 Step 3: Design nicking sgRNAs
21:30 Step 4: Identify helpful silent edits
24:04 Step 5: Select a PE system
27:35 Tips for optimizing parameters
39:16 Example applications
41:30 Summary and Acknowledgements
Q&A
43:57 Prime editing efficiency using other variants of Cas9
46:12 How does twinPE compare to PE3?
47:40 Do silent mutations have to be close to the target edit?
48:49 How do you design the RTT after including a silent edit?
49:22 Adding structural RNA motifs
50:56 Will PE6 approaches include CRISPRa-like strategies?
52:59 Minimizing off-target effects of prime editing
56:10 Using lipid nanoparticle delivery
57:22 Cloning pegRNA into a plasmid or synthesizing to transfect directly
58:37 Tips for successfully edited clones
59:37 Defining PEmax + comparing PEmax to PE2
1:01:14 How to make repeated sequential edits on the same locus
1:02:03 Tips for prime editing in plants
1:02:54 If non-spacer regions of pegRNA bind to genomic DNA
1:04:26 Flap exonucleases involved in prime editing
1:05:29 The largest insertion made using twinPE
1:07:08 How important is a linker region?
1:09:04 Impact of cell state on optimal extension design
1:10:27 Applications for insecticide resistance in mosquitos
❓ Share your question in the webinar Q&A ➡ bio-protocol.org/webinar/deta...
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🔧 Helpful prime editing resources:
Designing and executing prime editing experiments in mammalian cells
Doman, J.L., Sousa, A.A., Randolph, P.B. et al. Nat Protoc 17, 2431-2468 (2022): doi.org/10.1038/s41596-022-00...
Find constructs www.addgene.org/
Search-and-replace genome editing without double-strand breaks or donor DNA
Anzalone, A. V. et al. Nature 576, 149-157 (2019): doi.org/10.1038/s41586-019-17...
Engineered pegRNAs improve prime editing efficiency
Nelson, J. W. et al. Nat. Biotechnol. 40, 402-410 (2022): doi.org/10.1038/s41587-021-01...
Enhanced prime editing systems by manipulating cellular determinants of editing outcomes
Chen, P. J. et al. Cell 184, 5635-5652 (2021): doi.org/10.1016/j.cell.2021.0...
Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing
Anzalone, A. V. et al. Nat. Biotechnol. 40, 731-740 (2022): doi.org/10.1038/s41587-021-01...
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The Bio-protocol webinar series highlights technical advances in biomedical and life science, focusing on a detailed presentation of cutting-edge technologies and techniques. We aim to make this program a go-to place for sharing, learning, discussing, and uncovering how to apply new technologies and tools in the life science community.
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[@LakshmikanthanP-z9t]

Thanks for this webinar

[@flaviorump607]

Thanks for making this webinar, very useful!

[@ip2w715]

How does it work for deletions?

[@StevenChartrand-xo5iz]

what is the sgRNA or what is acting in its place because
isnt that from crispr?