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; Reti, Daniel; Wilson, Laurence O.W.; Bauer, Denis C.; Thomas, Paul Q.

doi:10.1101/2021.07.01.450810

Cited by 9 publications

(21 citation statements)

References 35 publications

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“…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: Introductionmentioning

confidence: 76%

“…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: Discussionmentioning

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%

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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

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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.

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Section: Introductionmentioning

confidence: 76%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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

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“…R1 Mouse embryonic stem cells were cultured and transfected as described by Adikusuma et al . (2021) 67 without modification.…”

Section: Methodsmentioning

confidence: 99%

“…Complementary gRNA oligos were designed with overhangs facilitating golden gate assembly and cloned into pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene plasmid #62988) according to Ran et al (2013) 64 . R1 Mouse embryonic stem cells were cultured and transfected as described by Adikusuma et al (2021) 66 without modification.…”

Section: Cell Culture and Transfectionmentioning

confidence: 99%

Leveraging a natural murine meiotic drive to suppress invasive populations

Gierus

Birand

Bunting

et al. 2022

Preprint

Self Cite

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Invasive rodents, including house mice, are a major cause of environmental damage and biodiversity loss, particularly in island ecosystems. Eradication can be achieved through the distribution of rodenticide, but this approach is expensive to apply at scale, can have negative impacts (e.g. on non-target species, or through contamination), has animal ethics concerns, and has restrictions on where it can be used. Gene drives, which exhibit biased inheritance, have been proposed as a next generation strategy to control invasive alien pests and disease vectors. However, synthetic gene drives including CRISPR homing drives have proven to be technically challenging to develop in mice. The t haplotype is a naturally-occurring segregation distortion locus with highly biased transmission from heterozygous males. Here we propose a novel gene drive strategy for population suppression, tCRISPR, that leverages t haplotype bias and an embedded SpCas9/gRNA transgene to spread inactivating mutations in a haplosufficient female fertility gene. Using spatially explicit individual-based in silico modelling, we show that polyandry, sperm competition, dispersal, and transmission bias are critical factors for tCRISPR-mediated population suppression. Modelling of realistic parameter values indicates that tCRISPR can eradicate an island population of 200,000 mice while the unmodified t haplotype fails under the same conditions. We also demonstrate feasibility of this approach by engineering tCRISPR mice in a safe split drive format. tCRISPR mice exhibit high transmission of the modified t haplotype, and efficient generation and transmission of inactivating mutations in a recessive female fertility gene, crucially, at levels for which the modelling predicts that population eradication can occur. This is the first example of a feasible gene drive system for invasive alien rodent population control.

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Precision genome editing in plants using gene targeting and prime editing: existing and emerging strategies

Hassan

Yuan

Liu

et al. 2022

Biotechnology Journal

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Precise modification of plant genomes, such as seamless insertion, deletion, or replacement of DNA sequences at a predefined site, is a challenging task. Gene targeting (GT) and prime editing are currently the best approaches for this purpose. However, these techniques are inefficient in plants, which limits their applications for crop breeding programs. Recently, substantial developments have been made to improve the efficiency of these techniques in plants. Several strategies, such as RNA donor templating, chemically modified donor DNA template, and tandem‐repeat homology‐directed repair, are aimed at improving GT. Additionally, improved prime editing gRNA design, use of engineered reverse transcriptase enzymes, and splitting prime editing components have improved the efficacy of prime editing in plants. These emerging strategies and existing technologies are reviewed along with various perspectives on their future improvement and the development of robust precision genome editing technologies for plants.

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

Cited by 9 publications

References 35 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

Leveraging a natural murine meiotic drive to suppress invasive populations

Precision genome editing in plants using gene targeting and prime editing: existing and emerging strategies

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