Designing and executing prime editing experiments in mammalian cells

Improve Research Reproducibility A Bio-protocol resource

Designing and executing prime editing experiments in mammalian cells

Speaker: Jordan Doman Moderator: Peyton Randolph

Online live: May 16, 2023 12:00 PM EST Views: 4054

Q&A

Abstract

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). PE 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 PE 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 (>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.

The speakers will discuss:

* Prime editing to precisely install or correct mutations in mammalian cells

* Optimization of prime editing strategies for a new edit

Speaker

Jordan Doman, Ph.D.

Graduate Student, Harvard University and the Broad Institute

Jordan Doman is a graduate student completing her PhD research in the laboratory of David R. Liu at the Broad Institute of MIT and Harvard. Her res...

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Moderator

Peyton Randolph, B.SC

Graduate Student, Harvard University and the Broad Institute

Peyton Randolph is a graduate student conducting doctoral research in David R. Liu's lab at the Broad Institute of MIT and Harvard. After assisting...

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References

Anzalone, A. V. et al. Nature 576, 149–157 (2019): https://doi.org/10.1038/s41586-019-1711-4

Nelson, J. W. et al. Nat. Biotechnol. 40, 402–410 (2022): https://doi.org/10.1038/s41587-021-01039-7

Chen, P. J. et al. Cell 184, 5635–5652 (2021): https://doi.org/10.1016/j.cell.2021.09.018

Anzalone, A. V. et al. Nat. Biotechnol. 40, 731–740 (2022): https://doi.org/10.1038/s41587-021-01133-w

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39 Q&A

How to access synthesized epegRNA?

I want to use epegRNA in synthesized RNA form. However, the companies providing RNA synthesis services usually synthesize RNA up to 150 nucleotides while epegRNAs are usually larger than 150 nucleotides. Moreover, epegRNA synthesized by in vitro transcription does not include appropriate modification such as phosphorthioate bonds and 2'OMe modification.Is there any option to access epegRNA with proper modification in synthesized RNA form?

1 Answer 25 Views May 4, 2023

Jordan Doman

When we want to use an epegRNA that is longer than the limit of RNA that companies can synthesize, we do remove the epegRNA motif to help decrease the length. Members of our lab have been able to obtain robust editing without the use of epegRNAs- see Chen et al 2021. As the synthesis capabilities of IDT and others expand, we hope that more epegRNA lengths will become accessible! We typically do not use in vitro transcription to generate epegRNAs because, as you mentioned, the lack of chemical modifications does decrease stability.

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