Abstract
Prime editor (PE), a versatile editor that allows the insertion and deletion of arbitrary sequences, and all 12-point mutations without double-strand breaks (DSB) and a donor template, dramatically enhances research capabilities. PE combines nickase Cas9(H840A) and reverse transcriptase (RT), along with prime editing guide RNA (pegRNA). It has been reported in several plant species, but a weak editing efficiency has led to a decrease in applications. This study reports an optimized-prime editor (O-PE) for endogenous gene editing in Arabidopsis thaliana cells, with an average 1.15% editing efficiency, which is 16.4-fold higher than previously reported. Meanwhile, we observed an increase in indels when testing alternative reverse transcriptase and found out that nCas9(H840A) fused to non-functional reverse transcriptase was responsible for the increase. This work develops an efficient prime editor for plant cells and provides a blueprint for applying PE in other photoautotrophic cells, such as microalgae, that have a high industrial value.
Highlights
Cas protein to introduce a double-strand break (DSB) at the target site. This was followed by the introduction of a donor template, along with a reliance on cell repair machinery, either homologues repair (HR) or non-homologues end-joining (NHEJ), for insertion or deletion of the sequence [2]
Prime editor (PE) consists of reverse transcriptase fused to nickase Cas9(H840A) via a flexible linker (Figure 1)
The editing efficiency of the PE system in different species cies is affected [26] by the selection of the spacer position, length of the primer binding site (PBS), size of the reverse transcriptase (RT)
Summary
Prime editor (PE), a versatile editor that allows the insertion and deletion of arbitrary sequences, and all 12-point mutations without double-strand breaks (DSB) and a donor template, dramatically enhances research capabilities. PE combines nickase Cas9(H840A) and reverse transcriptase (RT), along with prime editing guide RNA (pegRNA) It has been reported in several plant species, but a weak editing efficiency has led to a decrease in applications. Cas protein to introduce a double-strand break (DSB) at the target site This was followed by the introduction of a donor template, along with a reliance on cell repair machinery, either homologues repair (HR) or non-homologues end-joining (NHEJ), for insertion or deletion of the sequence [2]. A versatile editing tool, i.e., prime editor (PE), was reported, allowing insertion and deletion of an arbitrary sequence and all 12-point mutations, without DSB and a donor template [5]. NCas nicks the non-target strand, PBS binds to the 30 flap acting as a primer for RT, and transcribes the RT template containing the desired
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