Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1145555 | ENA |
REPOSITORIES: ENA
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Project description:The prime editing (PE) system consists of a Cas9 nickase fused to a reverse transcriptase, which introduces precise edits into the target genomic region guided by a prime editing guide RNA. However, PE efficiency is limited by mismatch repair. To overcome this limitation, transient expression of a dominant-negative MLH1 (MLH1dn) has been used to inhibit key components of mismatch repair. Here, we designed a de novo MLH1 small binder (MLH1-SB) that binds to the dimeric interface of MLH1 and PMS2 using RFdiffusion and AlphaFold 3. The compact size of MLH1-SB enabled its integration into existing PE architectures via 2A systems, creating a novel PE-SB platform. The PE7-SB system significantly improved PE efficiency, achieving an 18.8-fold increase over PEmax and a 2.5-fold increase over PE7 in HeLa cells, as well as a 3.4-fold increase over PE7 in mice. This study highlights the potential of generative AI in advancing genome editing technology.
2025-04-07 | GSE293633 | GEO
Project description:Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived human pluripotent stem cells (hPSCs). Unlike Cas9-mediated knock-in, cytosine base editor (CBE) and prime editor (PE) achieve the desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active DNA repair pathways and are particularly susceptible to p53-dependent cell death. These unique characteristics impede the efficiency of gene editing in hPSCs. Here, we demonstrate that dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by CBE or PE additively enhanced editing efficiency in hPSCs. The BE4stem system comprised of dominant negative p53 (p53DD) and three UNG inhibitor (UGI), engineered to specifically diminish base excision repair (BER), improved CBE efficiency in hPSCs. Addition of dominant negative MLH1 to inhibit mismatch repair activity and p53DD in the conventional PE system also significantly enhanced PE efficiency in hPSCs. Thus, combined inhibition of the unique cellular cascades engaged in hPSCs upon gene editing could significantly enhance precise genome editing in these cells.
2025-01-01 | GSE248190 | GEO
Project description:Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived human pluripotent stem cells (hPSCs). Unlike Cas9-mediated knock-in, cytosine base editor (CBE) and prime editor (PE) achieve the desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active DNA repair pathways and are particularly susceptible to p53-dependent cell death. These unique characteristics impede the efficiency of gene editing in hPSCs. Here, we demonstrate that dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by CBE or PE additively enhanced editing efficiency in hPSCs. The BE4stem system comprised of dominant negative p53 (p53DD) and three UNG inhibitor (UGI), engineered to specifically diminish base excision repair (BER), improved CBE efficiency in hPSCs. Addition of dominant negative MLH1 to inhibit mismatch repair activity and p53DD in the conventional PE system also significantly enhanced PE efficiency in hPSCs. Thus, combined inhibition of the unique cellular cascades engaged in hPSCs upon gene editing could significantly enhance precise genome editing in these cells.
2024-04-22 | GSE247589 | GEO
Project description:Prime editor (PE) has been recently developed to induce efficient and precise on-target editing, whereas its guide RNA (gRNA)-independent off-target effects remain unknown. Here, we used whole-genome and whole-transcriptome sequencing to determine gRNA-independent off-target mutations in cells expanded from single colonies, in which PE generated precise editing at on-target sites. We found that PE triggered no observable gRNA-independent off-target mutation genome-wide or transcriptome-wide in transfected human cells, highlighting its high specificity.
2022-02-03 | GSE178112 | GEO
Project description:Prime editor (PE) has been recently developed to induce efficient and precise on-target editing, whereas its guide RNA (gRNA)-independent off-target effects remain unknown. Here, we used whole-genome and whole-transcriptome sequencing to determine gRNA-independent off-target mutations in cells expanded from single colonies, in which PE generated precise editing at on-target sites. We found that PE triggered no observable gRNA-independent off-target mutation genome-wide or transcriptome-wide in transfected human cells, highlighting its high specificity.
2022-02-03 | GSE178111 | GEO
Project description:Prime editing is a highly versatile CRISPR-based genome editing technology with the potential to correct the vast majority of genetic defects1. However, correction of a disease phenotype in vivo in somatic tissues has not been achieved yet. Here, we establish proof-of-concept for in vivo prime editing, that resulted in rescue of a metabolic liver disease. We first develop a size-reduced prime editor (PE) lacking the RNaseH domain of the reverse transcriptase (SpCas9-PERnH), and a linker- and NLS-optimized intein-split PE construct (SpCas9-PE p.1153) for delivery by adeno-associated viruses (AAV). Systemic dual AAV-mediated delivery of this variant in neonatal mice enables installation of a transversion mutation at the Dnmt1 locus with 15% efficiency on average. Next, we targeted the disease-causing mutation in the phenylalanine hydroxylase (Pah)enu2 mouse model for phenylketonuria (PKU). Correction rates of 1.5% using the dual AAV approach could be increased to up to 14% by delivery of full-length SpCas9-PE via adenoviral vector 5 (AdV5), leading to full restoration of physiological blood phenylalanine (L-Phe) levels below 120 µmol/L. Our study demonstrates in vivo prime editing in the liver at two independent loci, emphasizing the potential of PEs for future therapeutic applications.
2021-12-29 | GSE174757 | GEO
Project description:Prime editor (PE) has wide application prospects in disease treatment due to its diversity of editing outcomes. However, the editing efficiency of PE still needs to be further improved for therapeutic applications. Here, we increase the probability of hybridization of the pegRNA primer binding site (PBS) to single-stranded DNA flap by adding additional PBS sequence and reverse transcription template (RTT) to the loop region of pegRNA. The selection of loop and the design of loop length are optimized. The resulting modified loop2 epegRNA (ML-epegRNA) showed higher prime editing efficiency than that of the epegRNA in HEK293T cells. In addition, we used ML-epegRNA for PE editing of multiple editing types in multiple cell lines, and the results showed a general improvement in editing efficiency. Overall, the ML-epegRNA expands the capabilities of genome editing tools.
2025-11-17 | GSE274737 | GEO
Project description:Purpose: The goals of this study are to introduce a new genome editing tool, which has the higher editing scope than the original genome editing tools. Methods: First, we transfected PE2 (the original prime editing tool, prime editor2), PE3 (the original prime editing tool, prime editor3) and HOPE (the new tool we developed in this study) vectors into human cells, respectively. Then, we harvested the genomic DNA form the transfected cells and amplified the specified amplicons. Finally, we used targeted amplicon sequencing approach to compare the editing efficiency and presion of the new tool with the original reported tools. Results: Our new genome editing tool improves the editing efficiency of prime editing without increasing the risk of undesired indels formation. Conclusions: We deleveped a new genome editing tool to increase the likelihood of successful gene engineering.
2021-04-10 | GSE171470 | GEO
Project description:Single nucleotide variants (SNVs) and small insertions or deletions (indels) underlie most rare monogenic disorders, yet therapeutic strategies to precisely correct these mutations remain limited. Prime Editing enables repair of such pathogenic variants without introducing double-stranded breaks. Here, we applied CRISPR Prime Editing to model and correct a de novo GDF11 nonsense mutation (Tyr336*) identified in a participant from the Undiagnosed Diseases Network (UDN) with growth delay and multisystem abnormalities. Using HEK293T cells, we generated heterozygous GDF11 Tyr336* clones, which exhibited reduced GDF11 protein levels due to post-translational degradation likely mediated by ER- and Golgi-associated quality control pathways. These cells displayed marked Golgi abnormalities, including an increased number of compact, irregularly shaped Golgi structures, findings consistent with Golgi fragmentation and stress. Transcriptomic profiling of HET cells revealed broad dysregulation of gene networks, including downregulation of metabolic and Golgi-linked biosynthetic genes, and upregulation of cell-adhesion and extracellular matrix genes. These transcriptional shifts paralleled the participant’s developmental, neural, and cardiovascular phenotypes. To correct the mutation, we tested multiple bespoke Prime Editing strategies and identified PE7, in combination with a pegRNA designed by Pridict, as the most effective ribonucleoprotein complex for rescue. Editing efficiency was further enhanced by introducing an additional silent PAM-disrupting mutation, likely preventing both Cas9 re-binding and mismatch repair. Together, these findings support a haploinsufficiency mechanism for the GDF11 Tyr336* allele and establish a generalizable framework for disease modeling and allele-specific correction of pathogenic variants in human cells.
2025-12-02 | GSE312163 | GEO