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Targeted insertion of large genetic payloads using cas directed LINE-1 reverse transcriptase.

ABSTRACT: 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.

SUBMITTER: Manoj F

PROVIDER: S-EPMC8654924 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Targeted insertion of large genetic payloads using cas directed LINE-1 reverse transcriptase.

Manoj Femila F Tai Laura W LW Wang Katelyn Sun Mi KSM Kuhlman Thomas E TE

Scientific reports 20211208 1

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 platfor ...[more]

PMID: 34880381

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