Enhancing prime editing by Csy4-mediated processing of pegRNA

Liu, Yao; Yang, Guang; Huang, Shuhong; Li, Xiangyang; Wang, Xin; Li, Guanglei; Tian, Chao; Chen, Yulin; Huang, Xingxu; Wang, Xiaolong

doi:10.1038/s41422-021-00520-x

Cited by 85 publications

(52 citation statements)

References 5 publications

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“…To overcome the problem of low prime editing, the use of Csy4 RNA endonuclease was applied to peg RNA (Fig. 1 h–k), [ 22 ]. First, a system capable of inducing independent expression of Csy4 RNA endonuclease and FnCas9(H969A)-RT was constructed (hereafter PE4 delivery method), then the pegRNA and ngRNA were linked through the Csy4 recognition site so that they could act simultaneously in the cell (Fig.…”

Section: Resultsmentioning

confidence: 99%

Expansion of the prime editing modality with Cas9 from Francisella novicida

Lee

Hur

et al. 2022

Genome Biol

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Prime editing can induce a desired base substitution, insertion, or deletion in a target gene using reverse transcriptase after nick formation by CRISPR nickase. In this study, we develop a technology that can be used to insert or replace external bases in the target DNA sequence by linking reverse transcriptase to the Francisella novicida Cas9, which is a CRISPR-Cas9 ortholog. Using FnCas9(H969A) nickase, the targeting limitation of existing Streptococcus pyogenes Cas9 nickase [SpCas9(H840A)]-based prime editing is dramatically extended, and accurate prime editing is induced specifically for the target genes in human cell lines.

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Section: Resultsmentioning

confidence: 99%

Expansion of the prime editing modality with Cas9 from Francisella novicida

Lee

Hur

et al. 2022

Genome Biol

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“… 31 Moreover, several important progresses have also been made in enhancing the activity of prime editor itself. Equipping the prime editor with strong nuclear localization signal, 32 modifying the 3′ engineered pegRNA to reduce self-complementing 33 or degradation, 34 and enhancing the RT with ssDNA binding domain 35 were all capable of improving prime editing. It is, therefore, expectable that the introduction of these findings to WT-PE system may also increase its editing efficiency.…”

Section: Discussionmentioning

confidence: 99%

WT-PE: Prime editing with nuclease wild-type Cas9 enables versatile large-scale genome editing

Tao

Wang

et al. 2022

Sig Transduct Target Ther

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Large scale genomic aberrations including duplication, deletion, translocation, and other structural changes are the cause of a subtype of hereditary genetic disorders and contribute to onset or progress of cancer. The current prime editor, PE2, consisting of Cas9-nickase and reverse transcriptase enables efficient editing of genomic deletion and insertion, however, at small scale. Here, we designed a novel prime editor by fusing reverse transcriptase (RT) to nuclease wild-type Cas9 (WT-PE) to edit large genomic fragment. WT-PE system simultaneously introduced a double strand break (DSB) and a single 3′ extended flap in the target site. Coupled with paired prime editing guide RNAs (pegRNAs) that have complementary sequences in their 3′ terminus while target different genomic regions, WT-PE produced bi-directional prime editing, which enabled efficient and versatile large-scale genome editing, including large fragment deletion up to 16.8 megabase (Mb) pairs and chromosomal translocation. Therefore, our WT-PE system has great potential to model or treat diseases related to large-fragment aberrations.

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“…Recently, several progresses have been made in improving the efficiency of prime editing. Equipping the prime editor with strong nuclear localization signal [19], 3′ engineered pegRNA [18,20] or ssDNA binding domain [21] significantly enhanced prime editing. Moreover, endogenous mismatch repair pathway has been shown to inhibit the installation of the desired edits, and depletion of this pathway also improved prime editing [22].…”

Section: Discussionmentioning

confidence: 99%

“…For example, in prime editing medicated single base conversions of RUNX1 site (+ 1), C to G, and C to T conversions were ~ 2 times more efficient than that of C to A. In addition, it has been shown that 3′ extension of pegRNA, in particular the PBS, is capable of base-pairing with the spacer, leading to reduced prime editing [ 18 ]. Therefore, it is also possible that specific triplets in the 3′ extensions may disturb the architecture of pegRNA, thereby reducing prime editing.…”

Section: Discussionmentioning

confidence: 99%

Random-PE: an efficient integration of random sequences into mammalian genome by prime editing

et al. 2021

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Prime editing (PE) enables efficiently targeted introduction of multiple types of small-sized genetic change without requiring double-strand breaks or donor templates. Here we designed a simple strategy to introduce random DNA sequences into targeted genomic loci by prime editing, which we named random prime editing (Random-PE). In our strategy, the prime editing guide RNA (pegRNA) was engineered to harbor random sequences between the primer binding sequence (PBS) and homologous arm (HA) of the reverse transcriptase templates. With these pegRNAs, we achieved efficient targeted insertion or substitution of random sequences with different lengths, ranging from 5 to 10, in mammalian cells. Importantly, the diversity of inserted sequences is well preserved. By fine-tuning the design of random sequences, we were able to make simultaneously insertions or substitutions of random sequences in multiple sites, allowing in situ evolution of multiple positions in a given protein. Therefore, these results provide a framework for targeted integration of random sequences into genomes, which can be redirected for manifold applications, such as in situ protospacer adjacent motif (PAM) library construction, enhancer screening, and DNA barcoding.

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Enhancing prime editing by Csy4-mediated processing of pegRNA

Cited by 85 publications

References 5 publications

Expansion of the prime editing modality with Cas9 from Francisella novicida

Expansion of the prime editing modality with Cas9 from Francisella novicida

WT-PE: Prime editing with nuclease wild-type Cas9 enables versatile large-scale genome editing

Random-PE: an efficient integration of random sequences into mammalian genome by prime editing

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