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PE editing efficiency is enhanced by the addition of structured RNA motifs to the 3′ terminus of pegRNAs a, Efficiency of PE3-mediated insertions of the FLAG epitope tag at the +1 editing position (insertion directly at the pegRNA-induced nick site) across multiple genomic loci in HEK293T cells using canonical pegRNAs (‘unmodified’), pegRNAs with either evopreQ1 or mpknot appended to the 3′ end of the PBS via an 8-nt linker or pegRNAs appended with only the 8-nt linker sequence. b, Summary of the fold change in PE editing efficiency relative to canonical pegRNAs of the indicated edit at various genomic loci upon addition of the indicated 3′ motif via an 8-nt linker or the addition of the linker alone. ‘Transversion’ denotes mutation of the +5 G•C to T•A at RUNX1, EMX1, VEGFA and DNMT1, the +1 C•G to T•A at RNF2 and the +1 T•A to A•T at HEK3, where the positive integer indicates the distance from the Cas9 nick site. ‘Deletion’ denotes a 15-bp deletion at the Cas9 nick site. Data summarized here are presented in c and Supplementary Fig. 4. The horizontal bars show the median values. c, Representative improvements in PE efficiency from appending either evopreQ1 (p) or mpknot (m) via an 8-nt linker to pegRNAs with varying template lengths (in nucleotides, indicated). d, Editing activities of canonical pegRNAs and modified pegRNAs across three genomic loci in HeLa cells, U2OS cells and K562 cells. Data and error bars in a, c and d indicate the mean and standard deviation of three independent biological replicates.
Source publication
Prime editing enables the installation of virtually any combination of point mutations, small insertions or small deletions in the DNA of living cells. A prime editing guide RNA (pegRNA) directs the prime editor protein to the targeted locus and also encodes the desired edit. Here we show that degradation of the 3′ region of the pegRNA that contain...
Similar publications
Prime Editing is a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based genome editing technique having promising potential in terms of reducing off target activity. It introduces fragments of DNA sequences into the target site using a guide RNA (gRNA) molecule, composed of both the sequence that is to be inserted into the targe...
Citations
... Paired Cas9 nuclease that generates DNA double-strand breaks (DSBs) can mediate genomic deletion with unintended mutations (Hsu et al. 2014). The newly developed prime editors (PEs) could be used to program deletions ranging from 5 to 80 bp with high efficiency and modest precision in human cells (Anzalone et al. 2019;Nelson et al. 2022). A pair of prime editing guide RNAs (pegRNAs) targeting the opposite DNA strands achieved precise sequence deletion between two nicks at human genomic sites (Anzalone et al. 2022;Choi et al. 2022). ...
Efficient and precise genomic deletion shows promise for investigating the function of proteins in plant research and enhancing agricultural traits. In this study, we tested the PRIME-Del (PDel) strategy using a pair of prime editing guide RNAs (pegRNAs) that targeted opposite DNA strands and achieved an average deletion efficiency of 55.8% for 60 bp fragment deletions at six endogenous targets. Moreover, as high as 84.2% precise deletion efficiency was obtained for a 2000 bp deletion at the OsGS1 site in transgenic rice plants. To add the bases that were unintentionally deleted between the two nicking sequences, we used the PDel/Syn strategy, which introduced multiple synonymous base mutations in the region that had to be patched in the RT template. The PDel/Syn strategy achieved an average of 58.1% deletion efficiency at six endogenous targets, which was higher than the PDel strategy. The strategies presented in this study contribute to achieving more accurate and flexible deletions in transgenic rice plants.
... We next verified whether CC-PE could be delivered flexibly and yield precise editing in vivo using dual AAVs. To maximize in vivo prime editing efficiency, we applied the RNA structural motif, evopreQ 1 , to modify the 3′ terminus of pegRNAs (EpegRNA), a method that had been reported to improve prime editing efficiency by enhancing pegRNA stability and preventing its degradation [18]. ...
Background
Prime editing enables precise base substitutions, insertions, and deletions at targeted sites without the involvement of double-strand DNA breaks or exogenous donor DNA templates. However, the large size of prime editors (PEs) hampers their delivery in vivo via adeno-associated virus (AAV) due to the viral packaging limit. Previously reported split PE versions provide a size reduction, but they require intricate engineering and potentially compromise editing efficiency.
Results
Herein, we present a simplified split PE named as CC-PE, created through non-covalent recruitment of reverse transcriptase to the Cas9 nickase via coiled-coil heterodimers, which are widely used in protein design due to their modularity and well-understood sequence-structure relationship. We demonstrate that the CC-PE maintains or even surpasses the efficiency of unsplit PE in installing intended edits, with no increase in the levels of undesired byproducts within tested loci amongst a variety of cell types (HEK293T, A549, HCT116, and U2OS). Furthermore, coiled-coil heterodimers are used to engineer SpCas9-NG-PE and SpRY-PE, two Cas9 variants with more flexible editing scope. Similarly, the resulting NG-CC-PE and SpRY-CC-PE also achieve equivalent or enhanced efficiency of precise editing compared to the intact PE. When the dual AAV vectors carrying CC-PE are delivered into mice to target the Pcsk9 gene in the liver, CC-PE enables highly efficient precise editing, resulting in a significant reduction of plasma low-density lipoprotein cholesterol and total cholesterol.
Conclusions
Our innovative, modular system enhances flexibility, thus potentially facilitating the in vivo applicability of prime editing.
... Therefore, optimization of pegRNAs would ideally include pairwise combinatorial testing of a large number of various PBS and RTT lengths. An additional challenge for library screening is that prime editing efficiencies (especially in cell types other than HEK293s) are generally much lower than those achieved with CRISPR nuclease or base editor technologies, even with recent advances that include pegRNA modifications and coexpression of a dominant-negative protein involved in DNA mismatch repair 8,9 . ...
... MOSAIC provides a simple and rapid method to do so, enabling the screening of large numbers of pegRNA designs without the need for cloning or for viral vector technologies. pegRNAs optimized by MOSAIC can be further enhanced for their prime editing efficiencies by combining them with various ngRNAs (PE3 strategy) or with other recent advances such as epegRNAs and co-expression of a dominant-negative DNA mismatch repair protein 8,9,19 . In sum, the development and availability of MOSAIC should broadly extend and enhance both the research and therapeutic applications of PE technology. ...
CRISPR prime editing offers unprecedented versatility and precision for the installation of genetic edits in situ . Here we describe the development and characterization of the Multiplexing Of Site-specific Alterations for In situ Characterization ( MOSAIC ) method, which leverages a non-viral PCR-based prime editing method to enable rapid installation of thousands of defined edits in pooled fashion. We show that MOSAIC can be applied to perform in situ saturation mutagenesis screens of: (1) the BCR-ABL1 fusion gene, successfully identifying known and potentially new imatinib drug-resistance variants; and (2) the IRF1 untranslated region (UTR), re-confirming non-coding regulatory elements involved in transcriptional initiation. Furthermore, we deployed MOSAIC to enable high-throughput, pooled screening of hundreds of systematically designed prime editing guide RNA ( pegRNA ) constructs for a large series of different genomic loci. This rapid screening of >18,000 pegRNA designs identified optimized pegRNAs for 89 different genomic target modifications and revealed the lack of simple predictive rules for pegRNA design, reinforcing the need for experimental optimization now greatly simplified and enabled by MOSAIC. We envision that MOSAIC will accelerate and facilitate the application of CRISPR prime editing for a wide range of high-throughput screens in human and other cell systems.
... Adding RNA secondary structures (such as evopreQ, G-quadruplex, Zika xrRNA) to the 3′ end of the pegRNA can enhance efficiency by improving its stability and stimulating reverse transcription [51][52][53] . Prime editing efficiency can also be increased by introducing synonymous mutations of the host genome sequence in the reverse transcriptase template 54,55 . ...
... Consequently, PEs offer high editing purity and target specificity. Recent studies have further increased prime editing efficiency through the engineering of PEs and the optimization of pegRNAs to increase expression, nuclear localization, and degradation resistance [55][56][57][58][59][60] . PEs are remarkably versatile methods for precise genome editing, facilitating functional genomics investigations of nearly all types of genetic variation with exceptional specificity. ...
Advances in sequencing technology have greatly increased our ability to gather genomic data, yet understanding the impact of genetic mutations, particularly variants of uncertain significance (VUSs), remains a challenge in precision medicine. The CRISPR‒Cas system has emerged as a pivotal tool for genome engineering, enabling the precise incorporation of specific genetic variations, including VUSs, into DNA to facilitate their functional characterization. Additionally, the integration of CRISPR‒Cas technology with sequencing tools allows the high-throughput evaluation of mutations, transforming uncertain genetic data into actionable insights. This allows researchers to comprehensively study the functional consequences of point mutations, paving the way for enhanced understanding and increasing application to precision medicine. This review summarizes the current genome editing tools utilizing CRISPR‒Cas systems and their combination with sequencing tools for functional genomics, with a focus on point mutations.
... Towards optimizing PE efficiencies, we leveraged engineered pegRNAs (epegRNAs) 23 . 103 ...
Understanding the effects of rare genetic variants remains challenging, both in coding and non-coding regions. While multiplexed assays of variant effect (MAVEs) have enabled scalable functional assessment of variants, established MAVEs are limited by either exogenous expression of variants or constraints of genome editing. Here, we introduce a pooled prime editing (PE) platform in haploid human cells to scalably assay variants in their endogenous context. We first optimized delivery of variants to HAP1 cells, defining optimal pegRNA designs and establishing a co-selection strategy for improved efficiency. We characterize our platform in the context of negative selection by testing over 7,500 pegRNAs targeting SMARCB1 for editing activity and observing depletion of highly active pegRNAs installing loss-of-function variants. We next assess variants in MLH1 via 6-thioguanine selection, assaying 65.3% of all possible SNVs in a 200-bp region spanning exon 10 and distinguishing LoF variants with high accuracy. Lastly, we assay 362 non-coding MLH1 variants across a 60 kb region in a single experiment, identifying pathogenic variants acting via multiple mechanisms with high specificity. Our analyses detail how filtering for highly active pegRNAs can facilitate both positive and negative selection screens. Accordingly, our platform promises to enable highly scalable functional assessment of human variants.
... However, indel frequencies ranging from 0.7 to 1.5 % were also observed [53]. Currently, several groups are working on advanced PE systems to improve the editing efficiency, while reducing unwanted indel outcomes [54][55][56][57][58]. ...
... Another strategy is that of inhibiting the mismatch repair system to improve the likelihood of the induced edit to be retained, obtained by the development of PE4, which was then combined with PE3 to obtain PE5, characterised by up to 2.5-fold increased editing efficiency over PE3 [111]. A final effort has been made to stabilise pegRNA, preventing intracellular degradation [171], which, in combination with PE5max, resulted in a 12-fold increase in efficiency compared to PE3 [111]. Finally, nuclease specificity and editing efficiency can be tested by assays relying on green fluorescent protein trapping, allowing the selection of edited cells and identifying the nuclease-mediated cleavage sites suitable for SCD studies [172]. ...
Recent advancements in gene editing illuminate new potential therapeutic approaches for Sickle Cell Disease (SCD), a debilitating monogenic disorder caused by a point mutation in the β-globin gene. Despite the availability of several FDA-approved medications for symptomatic relief, allogeneic hematopoietic stem cell transplantation
(HSCT) remains the sole curative option, underscoring a persistent need for novel treatments. This review delves into the growing field of gene editing, particularly the extensive research focused on curing haemoglobinopathies like SCD. We examine the use of techniques such as CRISPR-Cas9 and homology-directed repair, base editing,
and prime editing to either correct the pathogenic variant into a non-pathogenic or wild-type one or augment fetal haemoglobin (HbF) production. The article elucidates ways to optimize these tools for efficacious gene editing with minimal off-target effects and offers insights into their effective delivery into cells. Furthermore, we
explore clinical trials involving alternative SCD treatment strategies, such as LentiGlobin therapy and autologous HSCT, distilling the current findings. This review consolidates vital information for the clinical translation of gene editing for SCD, providing strategic insights for investigators eager to further the development of gene
editing for SCD.
... Recent studies therefore utilized rational design to increase the performance of prime editing. For example, pegRNAs have been protected from exonucleases by fusing structural motifs to the 3' end (epegRNAs) 3 , the cellular DNA mismatch repair machinery has been altered to favor selection of the introduced edit 4 , and codon usage as well as domain architecture of the PE have been optimized (PEmax) 4 . Furthermore, a recent study employed directed protein evolution using phage-assisted continuous evolution (PACE) to enhance the activity of different RT enzymes for prime editing 5 . ...
... Plasmids containing epegRNAs were created by ligation of the annealed spacer, scaffold, and 3' extension oligos into the BsaIdigested pU6-pegRNA-GG-acceptor (Addgene #13277), pU6-tevopreQ1-GG-acceptor (Addgene #174038) with Golden Gate assembly as previously described 1,3 . To generate intein-split PE plasmids, inserts were ordered as gBlocks from Integrated DNA Technologies (IDT) or amplified from pCMV-PEmax plasmids using PCR. ...
Prime editing is a highly versatile genome editing technology that enables the introduction of base substitutions, insertions, and deletions. However, compared to traditional Cas9 nucleases prime editors (PEs) are less active. In this study we use OrthoRep, a yeast-based platform for directed protein evolution, to enhance the editing efficiency of PEs. After several rounds of evolution with increased selection pressure, we identify multiple mutations that have a positive effect on PE activity in yeast cells and in biochemical assays. Combining the two most effective mutations – the A259D amino acid substitution in nCas9 and the K445T substitution in M-MLV RT – results in the variant PE_Y18. Delivery of PE_Y18, encoded on DNA, mRNA or as a ribonucleoprotein complex into mammalian cell lines increases editing rates up to 3.5-fold compared to PEmax. In addition, PE_Y18 supports higher prime editing rates when delivered in vivo into the liver or brain. Our study demonstrates proof-of-concept for the application of OrthoRep to optimize genome editing tools in eukaryotic cells.
... In addition, the cell's repair machinery favours the incorporation of the old, non-edit containing 5′ flap instead of the edit-containing 3' flap synthesised during the PE process. Having said that, PE is constantly being improved upon, yielding many new PE variants that increase the overall correction efficiencies [146][147][148][149]. ...
The monogenetic disease epidermolysis bullosa (EB) is characterised by the formation of extended blisters and lesions on the patient’s skin upon minimal mechanical stress. Causal for this severe condition are genetic mutations in genes, leading to the functional impairment, reduction, or absence of the encoded protein within the skin’s basement membrane zone connecting the epidermis to the underlying dermis. The major burden of affected families justifies the development of long-lasting and curative therapies operating at the genomic level. The landscape of causal therapies for EB is steadily expanding due to recent breakthroughs in the gene therapy field, providing promising outcomes for patients suffering from this severe disease. Currently, two gene therapeutic approaches show promise for EB. The clinically more advanced gene replacement strategy was successfully applied in severe EB forms, leading to a ground-breaking in vivo gene therapy product named beremagene geperpavec (B-VEC) recently approved from the US Food and Drug Administration (FDA). In addition, the continuous innovations in both designer nucleases and gene editing technologies enable the efficient and potentially safe repair of mutations in EB in a potentially permanent manner, inspiring researchers in the field to define and reach new milestones in the therapy of EB.
