Peptide fusion improves prime editing efficiency

Velimirovic, Minja; Zanetti, Larissa C; Shen, Max W.; Fife, James D.; Lin, Lin; Cha, Minsun; Akinci, Ersin; Barnum, Danielle; Yu, Tian; Sherwood, Richard I.

doi:10.1038/s41467-022-31270-y

Cited by 39 publications

(14 citation statements)

References 27 publications

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“…In cultured mammalian cells, PEmax improves editing efficiency over PE2 by up to threefold on average. Sherwood and co-workers also increased cellular PE2 expression upon tethering peptides to PE2 70 . Using a high-throughput approach called PepSEq to identify candidate peptide fusions, they designed an IN-PE2 editor construct that shows improvement in mouse and human cell types (Fig.…”

Section: Enhanced Prime Editor Effector Proteinsmentioning

confidence: 91%

“…Furthermore, Wang and co-workers designed extended pegRNAs containing a 3ʹ Csy4 recognition motif hairpin, which are likely to enhance prime editing through the same mechanism 77 . In their enhanced prime editing system (ePE), these extended pegRNAs are also fused to a nicking sgRNA and co-delivered with Csy4 nuclease, 70 , pegRNAs carrying the F + E crRNA scaffold 54,78 , and ePE pegRNAs 77 improve prime editor and pegRNA expression. epegRNAs 54 , ePE pegRNAs 77 , xr-pegRNAs 74 , G-PE pegRNAs 75 and sPE pegRNAs 76 reduce pegRNA degradation.…”

Section: Table 1 (Continued) | Prime Editor and Pegrna Architecturesmentioning

confidence: 99%

“…a, Variants of prime editors or prime editing guide RNAs (pegRNAs) that ultimately enhance the creation of the edited 3′ DNA flap. PEmax 53 , IN-PE270 , pegRNAs carrying the F + E crRNA scaffold54,78 , and ePE pegRNAs77 improve prime editor and pegRNA expression. epegRNAs54 , ePE pegRNAs77 , xr-pegRNAs74 , G-PE pegRNAs75 and sPE pegRNAs76 reduce pegRNA degradation.…”

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confidence: 99%

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Prime editing for precise and highly versatile genome manipulation

2022

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single-stranded DNA (ssDNA) at the target site, triggering deamination by the tethered ssDNA-specific deaminase enzyme. Cytosine base editors (CBEs) and adenine base editors (ABEs) contain deaminases that catalyse C•G-to-T•A and A•T-to-G•C changes, respectively [27][28][29][30][31] . C•G-to-G•C base editors (CGBEs) are similar to CBEs, but stimulate replacement of the deaminated cytosine base with guanine, albeit with lower typical efficiencies and product purities compared to CBEs and ABEs [33][34][35][36] . Compared to Cas nucleases, base editors exhibit substantially greater efficiency with few indel byproducts, and show far fewer undesired consequences of DSBs than nucleases in side-by-side comparisons 19,21,[23][24][25][26]37,38 .Because base editors deaminate within a small window of 4-5 nucleotides (nt) canonically, C or A nucleotides very close to the target C or A can also undergo conversion, resulting in 'bystander editing'. The application of base editors to make changes in the coding sequence of genes usually results in only synonymous mutations within a typical base editing activity window 39 owing to the frequency of transition mutations being silent in the genetic code. Nevertheless, undesired base editing of bystander nucleotides can be challenging to avoid in some cases. Base-editing activity is also restricted by the targeting scope of the Cas domain, which requires the presence of a protospacer adjacent motif (PAM) sequence at a specific distance range (typically 15 ± 2 nt) from the target nucleotide. Moreover, some base editors can induce off-target mutations in DNA and RNA [40][41][42] . Although engineering efforts have mitigated many of these drawbacks [43][44][45][46][47][48][49][50][51] , current base editors can only make six of the 12 possible types of point mutation, leaving many other classes of possible DNA edits, such as insertions, deletions and most transversions, beyond the reach of base editing.Motivated by the need for a precise and highly versatile geneediting technology, prime editing enables all types of DNA substitution, small insertions and small deletions to be installed at targeted sites in the genomes of living cells without directly forming DSBs 52 (Fig. 1c). Prime editing minimally requires a prime editor (PE) protein, which is typically a fusion of a nickase Cas9 (nCas9) and a reverse transcriptase (RT), along with a prime editing guide RNA (pegRNA), which specifies the target site for the edit and contains a programmable RNA template for the desired DNA sequence change. To mediate editing, PEs nick the target site on the genome and extend new DNA sequence from this nick using the pegRNA as a template (Fig. 2). This edited DNA strand is then incorporated into the genome through endogenous cellular processes that can be promoted by also nicking the non-edited strand 52 .Through this mechanism of directly rewriting target DNA, prime editing offers extraordinarily high versatility, editing purity and DNA target specificity in comparison to base editing and HDR with nucl...

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Section: Enhanced Prime Editor Effector Proteinsmentioning

confidence: 91%

Section: Table 1 (Continued) | Prime Editor and Pegrna Architecturesmentioning

confidence: 99%

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confidence: 99%

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Prime editing for precise and highly versatile genome manipulation

2022

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“…5 ). For the fusion of different peptides to PE2 25 , 26 , similar to GCN4, these peptides show a slight effect on PE system. Taken together, these studies appear contradictory but are consistent with our results.…”

Section: Discussionmentioning

confidence: 95%

“…Recent studies showed that the direct fusion of Rad51 DNA-binding domain 20 or various peptides 25 , 26 to PE2 generally increases the PE editing efficiencies. For the fusion of the Rad51 DNA-binding domain, the authors inserted Rad51 DBD into the region between nCas9 and RT domains, N- or C-terminus of PE2, generating variants named PE2-mid_Rad51, PE2-N_Rad51, and PE2-C_Rad5, respectively.…”

Section: Discussionmentioning

confidence: 99%

Enhancement of a prime editing system via optimal recruitment of the pioneer transcription factor P65

et al. 2023

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Prime editing is a versatile gene editing tool that enables precise sequence changes of all types in the genome, but its application is rather limited by the editing efficiency. Here, we first apply the Suntag system to recruit the transcription factor P65 and enhance the desired editing outcomes in the prime editing system. Next, MS2 hairpins are used to recruit MS2-fused P65 and confirmed that the recruitment of the P65 protein could effectively improve the prime editing efficiency in both the PE3 and PE5 systems. Moreover, this suggests the increased editing efficiency is most likely associated with the induction of chromatin accessibility change by P65. In conclusion, we apply different systems to recruit P65 and enhance the prime editing efficiency of various PE systems. Furthermore, our work provides a variety of methods to work as protein scaffolds for screening target factors and thus supports further optimization of prime editing systems.

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Integrating Prime Editing and Cellular Reprogramming as Novel Strategies for Genetic Cardiac Disease Modeling and Treatment

Yao,

Lei,

Gonçalves

et al. 2024

Curr Cardiol Rep

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Purpose of review This review aims to evaluate the potential of CRISPR-based gene editing tools, particularly prime editors (PE), in treating genetic cardiac diseases. It seeks to answer how these tools can overcome current therapeutic limitations and explore the synergy between PE and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) for personalized medicine. Recent findings Recent advancements in CRISPR technology, including CRISPR-Cas9, base editors, and PE, have demonstrated precise genome correction capabilities. Notably, PE has shown exceptional precision in correcting genetic mutations. Combining PE with iPSC-CMs has emerged as a robust platform for disease modeling and developing innovative treatments for genetic cardiac diseases. Summary The review finds that PE, when combined with iPSC-CMs, holds significant promise for treating genetic cardiac diseases by addressing their root causes. This approach could revolutionize personalized medicine, offering more effective and precise treatments. Future research should focus on refining these technologies and their clinical applications.

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Peptide fusion improves prime editing efficiency

Cited by 39 publications

References 27 publications

Prime editing for precise and highly versatile genome manipulation

Prime editing for precise and highly versatile genome manipulation

Enhancement of a prime editing system via optimal recruitment of the pioneer transcription factor P65

Integrating Prime Editing and Cellular Reprogramming as Novel Strategies for Genetic Cardiac Disease Modeling and Treatment

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