Efficient generation of mouse models with the prime editing system.
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ABSTRACT:
SUBMITTER: Liu Y
PROVIDER: S-EPMC7186222 | biostudies-literature | 2020
REPOSITORIES: biostudies-literature
Publications
Efficient generation of mouse models with the prime editing system.
Liu Yao Y Li Xiangyang X He Siting S Huang Shuhong S Li Chao C Chen Yulin Y Liu Zhen Z Huang Xingxu X Wang Xiaolong X
Cell discovery 20200428
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Project description:The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair of site-specific nuclease-induced double-strand breaks. Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs improved editing efficiencies up to 13-fold compared with transfecting the PE components as plasmids or ribonucleoprotein particles. Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson's disease in hPSCs.
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Project description:Using transient inhibition of DNA mismatch repair during a permissive stage of development, we demonstrate highly efficient prime editing of mouse embryos with few unwanted, local byproducts (average 58% precise edit frequency, 0.5% on-target error frequency across 13 substitution edits at 8 sites), enabling same-generation phenotyping of founders. Whole-genome sequencing reveals that mismatch repair inhibition increases off-target indels at low-complexity regions in the genome without any obvious phenotype in mice.
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Project description:Prime editing (PE) holds tremendous potential in the treatment of genetic diseases because it can install any desired base substitution or local insertion/deletion. However, the full-length PE effector size (6.3-kb) is beyond the packaging capacity of adeno-associated virus (AAV), hindering its clinical translation. Various splitting strategies have been used to improve its delivery, but always accompanied by compromised PE efficiency. Here, we developed a modular and efficient SunTag-PE system that splits PE effectors into GCN4-nCas9 and single-chain variable fragment (scFv) tethered reverse transcriptase (RT). We observed that SunTag-PEs with 1×GCN4 in the N terminus of nCas9 was the most efficient configuration rather than multiple copies of GCN4. This SunTag-PE strategy achieved editing levels comparable to canonical fused-PE (nCas9 and RT are linked together) and higher than other split-PE strategies (including sPE and MS2-PE) in both PE2 and PE3 forms with no increase in insertion and deletion (indel) byproducts. Moreover, we successfully validated the modularity of SunTag-PE system in the Cas9 orthologs of SauCas9 and FrCas9. Finally, we employed dual AAVs to deliver SunTag-ePE3 and efficiently corrected the pathogenic mutation in HBB mutant cell line. Collectively, our SunTag-PE system provides an efficient modular splitting strategy for prime editing and further facilitate its transformation in clinics.
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Project description:Realizing the promise of prime editing for the study and treatment of genetic disorders requires efficient methods for delivering prime editors (PEs) in vivo. Here we describe the identification of bottlenecks limiting adeno-associated virus (AAV)-mediated prime editing in vivo and the development of AAV-PE vectors with increased PE expression, prime editing guide RNA stability and modulation of DNA repair. The resulting dual-AAV systems, v1em and v3em PE-AAV, enable therapeutically relevant prime editing in mouse brain (up to 42% efficiency in cortex), liver (up to 46%) and heart (up to 11%). We apply these systems to install putative protective mutations in vivo for Alzheimer's disease in astrocytes and for coronary artery disease in hepatocytes. In vivo prime editing with v3em PE-AAV caused no detectable off-target effects or significant changes in liver enzymes or histology. Optimized PE-AAV systems support the highest unenriched levels of in vivo prime editing reported to date, facilitating the study and potential treatment of diseases with a genetic component.
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Project description:A recently developed adenine base editor (ABE) efficiently converts A to G and is potentially useful for clinical applications. However, its precision and efficiency in vivo remains to be addressed. Here we achieve A-to-G conversion in vivo at frequencies up to 100% by microinjection of ABE mRNA together with sgRNAs. We then generate mouse models harboring clinically relevant mutations at Ar and Hoxd13, which recapitulates respective clinical defects. Furthermore, we achieve both C-to-T and A-to-G base editing by using a combination of ABE and SaBE3, thus creating mouse model harboring multiple mutations. We also demonstrate the specificity of ABE by deep sequencing and whole-genome sequencing (WGS). Taken together, ABE is highly efficient and precise in vivo, making it feasible to model and potentially cure relevant genetic diseases.
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Project description:Prime editing is limited by low efficiency in plants. Here, we develop an upgraded engineered plant prime editor in hexaploid wheat, ePPEplus, by introducing a V223A substitution into reverse transcriptase in the ePPEmax* architecture. ePPEplus enhances the efficiency by an average 33.0-fold and 6.4-fold compared to the original PPE and ePPE, respectively. Importantly, a robust multiplex prime editing platform is established for simultaneous editing of four to ten genes in protoplasts and up to eight genes in regenerated wheat plants at frequencies up to 74.5%, thus expanding the applicability of prime editors for stacking of multiple agronomic traits.
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Project description:Prime editing is a recent genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. Here, we develop prime editing for primary adult stem cells grown in organoid culture models. First, we generate precise in-frame deletions in the gene encoding β-catenin (CTNNB1) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers disease-causing mutations in intestinal organoids from patients with DGAT1-deficiency and liver organoids from a patient with Wilson disease (ATP7B). Prime editing is as efficient in 3D grown organoids as in 2D grown cell lines and offers greater precision than Cas9-mediated homology directed repair (HDR). Base editing remains more reliable than prime editing but is restricted to a subgroup of pathogenic mutations. Whole-genome sequencing of four prime-edited clonal organoid lines reveals absence of genome-wide off-target effects underscoring therapeutic potential of this versatile and precise gene editing strategy.
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Project description:Retinitis pigmentosa (RP) is a complex spectrum of inherited retinal diseases marked by the gradual loss of photoreceptor cells, ultimately leading to blindness. Among these, mutations in PDE6A, responsible for encoding a cGMP-specific phosphodiesterase, stand out as pivotal in autosomal recessive RP (RP43). Unfortunately, no effective therapy currently exists for this specific form of RP. However, recent advancements in genome editing, such as base editing (BE) and prime editing (PE), offer a promising avenue for precise and efficient gene therapy. Here, it is illustrated that the engineered BE and PE systems, particularly PE, exhibit high efficiency in rescuing a target point mutation with minimal bystander effects in an RP mouse model carrying the Pde6a (c.2009A > G, p.D670G) mutation. The optimized BE and PE systems are first screened in N2a cells and subsequently assessed in electroporated mouse retinas. Notably, the optimal PE system, delivered via dual adeno-associated virus (AAV), precisely corrects the pathogenic mutation with average 9.4% efficiency, with no detectable bystander editing. This correction restores PDE6A protein expression, preserved photoreceptors, and rescued retinal function in Pde6a mice. Therefore, this study offers a proof-of-concept demonstration for the treatment of Pde6a-related retinal degeneration using BE and PE systems.
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