real-time-quantification-effects-is200is605-family-associated-tnpb-on

Real-Time Quantification of the Effects of IS200/IS605 Family-Associated TnpB on Transposon Activity

Here, a protocol is outlined to perform live, real-time imaging of transposable element activity in live bacterial cells using a suite of fluorescent ...

University of California

A difficult genome editing goal is the site-specific insertion of large genetic constructs. Here we describe the GENEWRITE system, where site-specific targetable activity of Cas endonucleases is coupled with the reverse transcriptase activity of the ORF2p protein of the human retrotransposon LINE-1. This is accomplished by providing two RNAs: a guide RNA targeting Cas endonuclease activity and an appropriately designed payload RNA encoding the desired insertion. Using E. coli as a simple platform for development and deployment, we show that with proper payload design and co-expression of helper proteins, GENEWRITE can enable insertion of large genetic payloads to precise locations, although with off-target effects, using the described approach. Based upon these results, we describe a potential strategy for implementation of GENEWRITE in more complex systems.

Femila Manoj

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JoVE Journal Publications

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