Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.
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ABSTRACT:
SUBMITTER: Chen PJ
PROVIDER: S-EPMC8584034 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Prime-editing systems have the capability to perform efficient and precise genome editing in human cells. In this study, we first developed a plant prime editor 2 (pPE2) system and test its activity by generating a targeted mutation on an HPT-ATG reporter in rice. Our results showed that the pPE2 system could induce programmable editing at different genome sites. In transgenic T0 plants, pPE2-generated mutants occurred with 0%-31.3% frequency, suggesting that the efficiency of pPE2 varied greatly at different genomic sites and with prime-editing guide RNAs of diverse structures. To optimize editing efficiency, guide RNAs were introduced into the pPE2 system following the PE3 and PE3b strategy in human cells. However, at the genomic sites tested in this study, pPE3 systems generated only comparable or even lower editing frequencies. Furthemore, we developed a surrogate pPE2 system by incorporating the HPT-ATG reporter to enrich the prime-edited cells. The nucleotide editing was easily detected in the resistant calli transformed with the surrogate pPE2 system, presumably due to the enhanced screening efficiency of edited cells. Taken together, our results indicate that plant prime-editing systems we developed could provide versatile and flexible editing in rice genome.
| S-EPMC7747961 | biostudies-literature
Project description:Precise genome editing in human pluripotent stem cells (hPSCs) has potential applications in isogenic disease modeling and ex vivo stem cell therapy, necessitating diverse genome editing tools. However, unlike differentiated somatic cells, hPSCs have unique cellular properties that maintain genome integrity, which largely determine the overall efficiency of an editing tool. Considering the high demand for prime editors (PEs), it is imperative to characterize the key molecular determinants of PE outcomes in hPSCs. Through homozygous knockout (KO) of MMR pathway key proteins MSH2, MSH3, and MSH6, we reveal that MutSα and MutSβ determine PE efficiency in an editing size-dependent manner. Notably, MSH2 perturbation disrupted both MutSα and MutSβ complexes, dramatically escalating PE efficiency from base mispair to 10 bases, up to 50 folds. Similarly, impaired MutSα by MSH6 KO improved editing efficiency from single to three base pairs, while defective MutSβ by MSH3 KO heightened efficiency from three to 10 base pairs. Thus, the size-dependent effect of MutSα and MutSβ on prime editing implies that MMR is a vital PE efficiency determinant in hPSCs and highlights the distinct roles of MutSα and MutSβ in its outcome.
| S-EPMC10280094 | biostudies-literature
Project description:CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) has been extensively exploited as a genetic tool for genome editing. The RNA guided Cas nucleases generate DNA double-strand break (DSB), triggering cellular repair systems mainly Non-homologous end-joining (NHEJ, imprecise repair) or Homology-directed repair (HDR, precise repair). However, DSB typically leads to unexpected DNA changes and lethality in some organisms. The establishment of bacteria and plants into major bio-production platforms require efficient and precise editing tools. Hence, in this review, we focus on the non-DSB and template-free genome editing, i.e., base editing (BE) and prime editing (PE) in bacteria and plants. We first highlight the development of base and prime editors and summarize their studies in bacteria and plants. We then discuss current and future applications of BE/PE in synthetic biology, crop improvement, evolutionary engineering, and metabolic engineering. Lastly, we critically consider the challenges and prospects of BE/PE in PAM specificity, editing efficiency, off-targeting, sequence specification, and editing window.
| S-EPMC7481536 | biostudies-literature
Project description:Prime editors (PEs) enable targeted precise editing, including the generation of substitutions, insertions and deletions, in eukaryotic genomes. However, their genome-wide specificity has not been explored. Here, we developed Nickase-based Digenome-seq (nDigenome-seq), an in vitro assay that uses whole-genome sequencing to identify single-strand breaks induced by CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) nickase. We used nDigenome-seq to screen for potential genome-wide off-target sites of Cas9 H840A nickase, a PE component, targeted to nine human genomic sites. Then, using targeted amplicon sequencing of off-target candidates identified by nDigenome-seq, we showed that only five off-target sites showed detectable PE-induced modifications in cells, at frequencies ranging from 0.1 to 1.9%, suggesting that PEs provide a highly specific method of precise genome editing. We also found that PE specificity in human cells could be further improved by incorporating mutations from engineered Cas9 variants, particularly eSpCas9 and Sniper Cas9, into PE.
| S-EPMC7544197 | biostudies-literature
Project description:Prime editing is a versatile and precise genome editing technique that can directly copy desired genetic modifications into target DNA sites without the need for donor DNA. This technique holds great promise for the analysis of gene function, disease modeling, and the correction of pathogenic mutations in clinically relevant cells such as human pluripotent stem cells (hPSCs). Here, we comprehensively tested prime editing in hPSCs by generating a doxycycline-inducible prime editing platform. Prime editing successfully induced all types of nucleotide substitutions and small insertions and deletions, similar to observations in other human cell types. Moreover, we compared prime editing and base editing for correcting a disease-related mutation in induced pluripotent stem cells derived form a patient with α 1-antitrypsin (A1AT) deficiency. Finally, whole-genome sequencing showed that, unlike the cytidine deaminase domain of cytosine base editors, the reverse transcriptase domain of a prime editor does not lead to guide RNA-independent off-target mutations in the genome. Our results demonstrate that prime editing in hPSCs has great potential for complementing previously developed CRISPR genome editing tools.
| S-EPMC8789035 | biostudies-literature
Project description:BackgroundThe CRISPR/Cas9 system, composed of a single-guide RNA for target recognition and a Cas9 protein for DNA cleavage, has the potential to revolutionize agriculture as well as medicine. Even though extensive work has been done to improve the gene editing activity of CRISPR/Cas9, little is known about the regulation of this bacterial system in eukaryotic host cells, especially at the post-transcriptional level.ResultsHere, we evaluate the expression levels of the two CRISPR/Cas9 components and the gene editing efficiency in a set of Arabidopsis mutants involved in RNA silencing. We find that mutants defective in the post-transcriptional gene-silencing pathway display significantly higher Cas9 and sgRNA transcript levels, resulting in higher mutagenesis frequencies than wild-type controls. Accordingly, silencing of AGO1 by introduction of an AGO1-RNAi cassette into the CRISPR/Cas9 vector provides an increase in gene editing efficiency. Co-expression of the viral suppressor p19 from the tomato bushy stunt virus to suppress the plant RNA-silencing pathway shows a strong correlation between the severity of the phenotypic effects caused by p19 and the gene editing efficiency of the CRISPR/Cas9 system for two different target genes, AP1 and TT4.ConclusionsThis system has useful practical applications in facilitating the detection of CRISPR/Cas9-induced mutations in T1 plants as well as the identification of transgene-free T2 plants by simple visual observation of the symptom severity caused by p19. Our study shows that CRISPR/Cas9 gene editing efficiency can be improved by reducing RNA silencing in plants.
| S-EPMC6161460 | biostudies-literature
Project description:Many genetic diseases and undesirable traits are due to base-pair alterations in genomic DNA. Base-editing, the newest evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based technologies, can directly install point-mutations in cellular DNA without inducing a double-strand DNA break (DSB). Two classes of DNA base-editors have been described thus far, cytosine base-editors (CBEs) and adenine base-editors (ABEs). Recently, prime-editing (PE) has further expanded the CRISPR-base-edit toolkit to all twelve possible transition and transversion mutations, as well as small insertion or deletion mutations. Safe and efficient delivery of editing systems to target cells is one of the most paramount and challenging components for the therapeutic success of BEs. Due to its broad tropism, well-studied serotypes, and reduced immunogenicity, adeno-associated vector (AAV) has emerged as the leading platform for viral delivery of genome editing agents, including DNA-base-editors. In this review, we describe the development of various base-editors, assess their technical advantages and limitations, and discuss their therapeutic potential to treat debilitating human diseases.
| S-EPMC7503568 | biostudies-literature
Project description:Inherited retinal diseases (IRDs) are chronic, hereditary disorders that lead to progressive degeneration of the retina. Disease etiology originates from a genetic mutation-inherited or de novo-with a majority of IRDs resulting from point mutations. Given the plethora of IRDs, to date, mutations that cause these dystrophies have been found in approximately 280 genes. However, there is currently only one FDA-approved gene augmentation therapy, Luxturna (voretigene neparvovec-rzyl), available to patients with RPE65-mediated retinitis pigmentosa (RP). Although clinical trials for other genes are underway, these techniques typically involve gene augmentation rather than genome surgery. While gene augmentation therapy delivers a healthy copy of DNA to the cells of the retina, genome surgery uses clustered regularly interspaced short palindromic repeats (CRISPR)-based technology to correct a specific genetic mutation within the endogenous genome sequence. A new technique known as prime editing (PE) applies a CRISPR-based technology that possesses the potential to correct all twelve possible transition and transversion mutations as well as small insertions and deletions. EDIT-101, a CRISPR-based therapy that is currently in clinical trials, uses double-strand breaks and nonhomologous end joining to remove the IVS26 mutation in the CEP290 gene. Preferably, PE does not cause double-strand breaks nor does it require any donor DNA repair template, highlighting its unparalleled efficiency. Instead, PE uses reverse transcriptase and Cas9 nickase to repair mutations in the genome. While this technique is still developing, with several challenges yet to be addressed, it offers promising implications for the future of IRD treatment.
| S-EPMC8656220 | biostudies-literature
Project description:Prime editing enables search-and-replace genome editing but is limited by low editing efficiency. We present a high-throughput approach, the Peptide Self-Editing sequencing assay (PepSEq), to measure how fusion of 12,000 85-amino acid peptides influences prime editing efficiency. We show that peptide fusion can enhance prime editing, prime-enhancing peptides combine productively, and a top dual peptide-prime editor increases prime editing significantly in multiple cell lines across dozens of target sites. Top prime-enhancing peptides function by increasing translation efficiency and serve as broadly useful tools to improve prime editing efficiency. Prime editing enables search-and-replace genome editing but is limited by low editing efficiency. Here the authors present PepSEq, a high-throughput method for screening a large library of peptides that influence prime editing efficiency.
| S-EPMC9206660 | biostudies-literature
Project description:Recent advances in CRISPR-Cas9 techniques, especially the discovery of base and prime editing, have significantly improved our ability to make precise changes in the genome. We hypothesized that modulating certain endogenous pathway cells could improve the action of those editing tools in mammalian cells. We established a reporter system in which a small fragment was integrated into the genome by prime editing (PE). With this system, we screened an in-house small-molecule library and identified a group of histone deacetylase inhibitors (HDACi) increasing prime editing. We also found that HDACi increased the efficiency of both cytosine base editing (CBE) and adenine base editing (ABE). Moreover, HDACi increased the purity of cytosine base editor products, which was accompanied by an upregulation of the acetylation of uracil DNA glycosylase (UNG) and UNG inhibitor (UGI) and an enhancement of their interaction. In summary, our work demonstrated that HDACi improves Cas9-mediated prime editing and base editing.
| S-EPMC9207553 | biostudies-literature