PEAR: a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells

Da, Simon; Tálas, András; Pi, Kulcsár; Welker, Ervin

doi:10.1101/2021.04.26.441486

Cited by 4 publications

(9 citation statements)

References 45 publications

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“…2b). SuperFi in combination with a prime editor was unsuccessful, as it showed little to no activity in the PEAR 33 fluorescent assay (Supplementary Fig. 7a) and showed no activity at all on 10 genomic loci installing 13 types of edits (Fig.…”

Section: Resultsmentioning

confidence: 99%

SuperFi-Cas9 exhibits extremely high fidelity but reduced activity in mammalian cells

Kulcsár

Tálas

Welker

2022

Preprint

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Several advances have been made to SpCas9, the most widely used CRISPR/Cas genome editing tool, to reduce its unwanted off-target effects. The most promising approach is the development of increased fidelity nuclease (IFN) variants of SpCas9, however, their fidelity has increased at the cost of reduced activity. SuperFi-Cas9 has been developed recently, and it has been described as a next-generation high fidelity SpCas9 variant, free from the drawbacks of the first-generation IFNs. In this study, we characterized the on-target activity and the off-target propensity of SuperFi-Cas9 in mammalian cells comparing it to first-generation IFNs. SuperFi-Cas9 demonstrated strongly reduced activity but exceptionally high fidelity exhibiting features that are in many aspects similar to those of the first-generation variants, such as evo- and HeFSpCas9. When combined with ABE8e, SuperFi-Cas9 produced DNA editing with high activity rate as well as high specificity by reducing both bystander and SpCas9-dependent off-target base editing.

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Section: Resultsmentioning

confidence: 99%

SuperFi-Cas9 exhibits extremely high fidelity but reduced activity in mammalian cells

Kulcsár

Tálas

Welker

2022

Preprint

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“…Typically, the most efficient strategy for the selection of edited cells is targeting endogenous genes as opposed to the use of exogenous markers 17,20,21,62 . Nonetheless, alternative methods have been described to enrich for edited cells, such as puromycin-based selection strategy and the use of surrogate reporters 14,16 .…”

Section: Discussionmentioning

confidence: 99%

“…Selecting cells that express all PE components with a selection marker is not sufficient for robust enrichment, while FACS-based cell sorting using exogenous gene markers could be prohibitive for some cell types due to cellular stress 14,16 .…”

Section: Discussionmentioning

confidence: 99%

“…All PCR amplifications were performed with 30 cycles of amplification. Sanger sequencing was performed on PCR amplicons to quantify the percentage of edited alleles using BEAT, TIDE, and TIDER 16,17,21 . Phusion and Q5 high-fidelity DNA polymerases were used for PCR amplification.…”

Section: Genotypingmentioning

confidence: 99%

“…This strategy yielded the PE4 (PE2+MLH1dn), PE5 (PE3+MLH1dn), and PE5b (PE3b+MLH1dn) editors 7 . Thus, the optimal PE strategy to adopt varies according to the context and there is a need to develop methods to consistently improve its success rate.To circumvent these limitations several approaches have been implemented including editing with purified ribonucleoprotein complexes (RNPs) 10 , mRNA-based delivery 7,11 , engineered pegRNAs (epegRNAs) 12 , NLS-and codon-optimized prime editors 7,9,13 , enrichment with puromycin, and fluorescent reporter-based selection [14][15][16] . Nevertheless, these improvements are still dependent on extensive pegRNA optimization, absolute activity remains low, and the streamlined production of homozygous cell lines has yet to be achieved 1,12 .…”

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confidence: 99%

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Marker-free coselection for successive rounds of prime editing in human cells

Levesque

Mayorga

Fiset

et al. 2021

Preprint

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Prime editing enables the introduction of precise point mutations, small insertions, or short deletions without requiring donor DNA templates. However, editing efficiency remains a key challenge in a broad range of cell types. We designed a robust coselection strategy to perform successive rounds of genetic changes in human cells using CRISPR-Cas nucleases and prime editors. Through coediting, the recovery of cells modified at a target gene of interest is coupled to selection for dominant alleles of the ubiquitous and essential sodium/potassium pump (Na+/K+ ATPase). The method improves prime editing efficiency 1.5–10-fold in K562 and HeLa cells, reaching up to 83% of desired alleles in cell populations, for previously optimized prime editing guide RNAs (pegRNAs). Using pegRNAs designed de novo, we readily engineered homozygous cell lines with cancer-associated or drug resistant MTOR mutations displaying altered mTORC1 signaling. Our approach streamlines the production of cell lines with multiple genetic modifications to create cellular models for biological research and lays the foundation for the development of cell-type specific coselection strategies.

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Mutation-specific reporter for optimization and enrichment of prime editing

et al. 2022

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Prime editing is a versatile genome-editing technique that shows great promise for the generation and repair of patient mutations. However, some genomic sites are difficult to edit and optimal design of prime-editing tools remains elusive. Here we present a fluorescent prime editing and enrichment reporter (fluoPEER), which can be tailored to any genomic target site. This system rapidly and faithfully ranks the efficiency of prime edit guide RNAs (pegRNAs) combined with any prime editor variant. We apply fluoPEER to instruct correction of pathogenic variants in patient cells and find that plasmid editing enriches for genomic editing up to 3-fold compared to conventional enrichment strategies. DNA repair and cell cycle-related genes are enriched in the transcriptome of edited cells. Stalling cells in the G1/S boundary increases prime editing efficiency up to 30%. Together, our results show that fluoPEER can be employed for rapid and efficient correction of patient cells, selection of gene-edited cells, and elucidation of cellular mechanisms needed for successful prime editing.

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PEAR: a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells

Cited by 4 publications

References 45 publications

SuperFi-Cas9 exhibits extremely high fidelity but reduced activity in mammalian cells

SuperFi-Cas9 exhibits extremely high fidelity but reduced activity in mammalian cells

Marker-free coselection for successive rounds of prime editing in human cells

Mutation-specific reporter for optimization and enrichment of prime editing

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