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.
Highlights
A difficult genome editing goal is the site-specific insertion of large genetic constructs
This is accomplished by coupling the targetable endonuclease activity of Cas enzymes to the reverse transcriptase activity of the human retrotransposon LINE-1 through translationally fusing Cas and LINE-1 reverse transcriptase proteins (Fig. 1A)
Previous studies have shown that reverse transcription by the LINE-1 protein ORF2p can be directed to pre-existing nicks and cuts in targeted DNA sequences in vitro[48], and we have previously shown that LINE-1 is functional in E. coli, when complemented by expression of enzymes for nonhomologous end joining (NHEJ) r epair[49]
Summary
A difficult genome editing goal is the site-specific insertion of large genetic constructs. We introduce a method for the active insertion of lengthy genetic sequences into host DNA we call GENEWRITE: Genome Engineering With RNA-Integrating Targetable Endonucleases This is accomplished by coupling the targetable endonuclease activity of Cas enzymes to the reverse transcriptase activity of the human retrotransposon LINE-1 through translationally fusing Cas and LINE-1 reverse transcriptase proteins (Fig. 1A). Insertion of these 2–3 kbp bacterial transposons is programmable to specific genomic locations in E. coli through a guide RNA similar to other Cas enzymes Another approach, prime editing[43], fuses a catalytically impaired Cas[9] fused to an engineered Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase[44,45,46,47], using a “prime editing guide RNA” (pegRNA) to target short insertions, deletions, and all types of point mutations into human cells. We illustrate the sitespecific reverse transcription and insertion of ~ 1.5 kbp payload RNAs, larger than that offered by prime editing
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