Optimized nickase- and nuclease-based prime editing in human and mouse cells

Adikusuma, Fatwa; Lushington, Caleb; Arudkumar, Jayshen; Godahewa, G.I.; Chey, Yu Chinn Joshua; Gierus, Luke; Piltz, Sandra; Geiger, Ashleigh; Jain, Yatish; Reti, Daniel; Wilson, Laurence O.W.; Bauer, Denis C.; Thomas, Paul Q.

doi:10.1093/nar/gkab792

Cited by 61 publications

(37 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The pAIO-PE3 plasmid, combined with a puromycin selection showed a significant increase compared to the regular PE3 system in mouse stem embryonic stem cells, with edit efficiencies ranging between 1.7% and 85%. In line with other ncRNA-dependent PE systems, pAIO-PE3 activity however also resulted in a high rate of indel/SNVgeneration for most loci, occurring in up to 90% of the clones [6] . Since PE3 systems are accompanied by higher undesired indels/SNVs, improved versions of ncRNA-independent PE systems would be safer alternatives for gene-editing in iPSC-derived models.…”

Section: Introductionsupporting

confidence: 70%

“…The few studies that tested multi-plasmid PE3 for editing in hiPSC showed a nearly undetectable edit efficiency [4] , making it not suitable for the generation of iPSC-derived disease models. However, the recently published PEA1 (pAIO-PE3) all-in-one plasmid was able to increase the edit efficiency significantly in immortalized cell lines as well as mouse embryonic stem cells (mESC) when combined with a puromycin selection step [6] . It did however induce similarly high unintended alterations as seen in the multi-plasmid approach.…”

Section: Pe4max and The Advantage Of Specific Base Changesmentioning

confidence: 99%

“…However, introducing or correcting mutations is still highly laborious and costly, creating a bottleneck for more high-throughput projects. Recently, an all-in-one plasmid (pAIO) was published that contains the complete PE3 system (PEA1) thus facilitating transfection of stem cells with all the necessary constructs at once [6] . The pAIO-PE3 plasmid, combined with a puromycin selection showed a significant increase compared to the regular PE3 system in mouse stem embryonic stem cells, with edit efficiencies ranging between 1.7% and 85%.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Efficient and accurate prime editing strategy to correct genetic alterations in hiPSC using single EF-1alpha driven all-in-one plasmids

Weuring

Dirkx

Vriendt

et al. 2022

Preprint

View full text Add to dashboard Cite

Prime editing (PE) is currently the most effective and versatile technology to make targeted alterations in the genome. Several improvements to the PE machinery have recently been made, and have been tested in a range of model systems, including immortalized cell lines, stem-cells and animal models. While nick RNA (ncRNA)-dependent PE systems like PE3 and PE5 are currently considered to be the most effective, they come with undesired indels or SNVs at the edit locus. Here, we aimed to improve ncRNA-independent systems PE2 and PE4max by generating novel all-in-one (pAIO) plasmids, driven by a tissue-broad EF-1alpha promoter, that is especially suitable for human iPSC models, and linked to a GFP tag for fluorescent based sorting. These novel pAIO systems effectively corrected mutations observed in patients suffering from epileptic encephalopathy, including a truncating SCN1A R612* variant in HEK293T cells and a gain-of-function KCNQ2 R201C variant in patient-derived hiPSC, with edit efficiency up to 50%. We also show that introducing additional silent PAM-removing mutations can negatively influence edit efficiency. Finally, we observed an absence of genome-wide PE off-target effects at pegRNA homology sites. Taken together, our study shows an improved efficacy and accuracy of EF-1alpha driven ncRNA-independent pAIO PE plasmids in hiPSC.

show abstract

Section: Introductionsupporting

confidence: 70%

Section: Pe4max and The Advantage Of Specific Base Changesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Efficient and accurate prime editing strategy to correct genetic alterations in hiPSC using single EF-1alpha driven all-in-one plasmids

Weuring

Dirkx

Vriendt

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…This strategy yielded the PE4 (PE2 + MLH1dn), PE5 (PE3 + MLH1dn), and PE5b (PE3b + MLH1dn) editors 7 . In addition, nuclease prime editors have been shown to improve PE efficiency, but it comes at the expense of product purity 10 – 12 . 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.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

View full text Add to dashboard Cite

Prime editing enables the introduction of precise point mutations, small insertions, or short deletions without requiring donor DNA templates. However, efficiency remains a key challenge in a broad range of human cell types. In this work, we design a robust co-selection strategy through coediting of the ubiquitous and essential sodium/potassium pump (Na+/K+ ATPase). We readily engineer highly modified pools of cells and clones with homozygous modifications for functional studies with minimal pegRNA optimization. This process reveals that nicking the non-edited strand stimulates multiallelic editing but often generates tandem duplications and large deletions at the target site, an outcome dictated by the relative orientation of the protospacer adjacent motifs. 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 co-selection strategies.

show abstract

“…We considered whether enrichment of genomic editing was caused by higher co-transfection rates of all prime editing plasmids in fluoPEER-edited cells 23 . We therefore transfected a mix of up to four fluorescent plasmids (mTurq2, eGFP, mKO2, mCherry) and tested co-transfection efficiency using FACS.…”

Section: Resultsmentioning

confidence: 99%

Mutation-specific reporter for optimization and enrichment of prime editing

et al. 2022

View full text Add to dashboard Cite

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.

show abstract

Optimized nickase- and nuclease-based prime editing in human and mouse cells

Cited by 61 publications

References 30 publications

Efficient and accurate prime editing strategy to correct genetic alterations in hiPSC using single EF-1alpha driven all-in-one plasmids

Efficient and accurate prime editing strategy to correct genetic alterations in hiPSC using single EF-1alpha driven all-in-one plasmids

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

Mutation-specific reporter for optimization and enrichment of prime editing

Contact Info

Product

Resources

About