Enhancing prime editing efficiency by modified pegRNA with RNA G-quadruplexes.
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ABSTRACT:
SUBMITTER: Li X
PROVIDER: S-EPMC9345309 | biostudies-literature | 2022 Aug
REPOSITORIES: biostudies-literature
Publications
Enhancing prime editing efficiency by modified pegRNA with RNA G-quadruplexes.
Li Xiangyang X Wang Xin X Sun Wenjun W Huang Shisheng S Zhong Mingtian M Yao Yuan Y Ji Quanjiang Q Huang Xingxu X
Journal of molecular cell biology 20220801 4
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Project description:CRISPR prime editing (PE) requires a Cas9 nickase-reverse transcriptase fusion protein (known as PE2) and a prime editing guide RNA (pegRNA), an extended version of a standard guide RNA (gRNA) that both specifies the intended target genomic sequence and encodes the desired genetic edit. Here we show that sequence complementarity between the 5' and the 3' regions of a pegRNA can negatively impact its ability to complex with Cas9, thereby potentially reducing PE efficiency. We demonstrate this limitation can be overcome by a simple pegRNA refolding procedure, which improved ribonucleoprotein-mediated PE efficiencies in zebrafish embryos by up to nearly 25-fold. Further gains in PE efficiencies of as much as 6-fold could also be achieved by introducing point mutations designed to disrupt internal interactions within the pegRNA. Our work defines simple strategies that can be implemented to improve the efficiency of PE.
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Project description:Prime editors are CRISPR-based genome engineering tools with significant potential for rectifying patient mutations. However, their usage requires experimental optimization of the prime editing guide RNA (PegRNA) to achieve high editing efficiency. This paper introduces the deep transformer-based model for predicting prime editing efficiency (DTMP-Prime), a tool specifically designed to predict PegRNA activity and prime editing (PE) efficiency. DTMP-Prime facilitates the design of appropriate PegRNA and ngRNA. A transformer-based model was constructed to scrutinize a wide-ranging set of PE data, enabling the extraction of effective features of PegRNAs and target DNA sequences. The integration of these features with the proposed encoding strategy and DNABERT-based embedding has notably improved the predictive capabilities of DTMP-Prime for off-target sites. Moreover, DTMP-Prime is a promising tool for precisely predicting off-target sites in CRISPR experiments. The integration of a multi-head attention framework has additionally improved the precision and generalizability of DTMP-Prime across various PE models and cell lines. Evaluation results based on the Pearson and Spearman correlation coefficient demonstrate that DTMP-Prime outperforms other state-of-the-art models in predicting the efficiency and outcomes of PE experiments.
| S-EPMC11626815 | biostudies-literature
Project description:The prime editor is a versatile tool for targeted precise editing to generate point mutations, small insertions, or small deletions in eukaryotes. However, canonical PE3 system is less efficient, notably in primary cells or pluripotent stem cells. Here, we employed RNA polymerase II promoter instead of RNA polymerase III promoter, whose application is limited by specific DNA contexts, to produce Csy4-processed intronic prime editing guide RNAs (pegRNAs) and, together with other optimizations, achieved efficient targeting with poly(T)-containing pegRNAs, as well as combinatorial and conditional genetic editing. We also found simultaneous suppression of both DNA mismatch repair and DNA damage response could achieve efficient and accurate editing in human embryonic stem cells. These findings relieve the restrictions of RNA polymerase III (RNA-Pol-III)-based base editors and broadened the applications of prime editing.
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Project description:Prime editing systems have enabled the incorporation of precise edits within a genome without introducing double strand breaks. Previous studies defined an optimal primer binding site (PBS) length for the pegRNA of ∼13 nucleotides depending on the sequence composition. However, optimal PBS length characterization has been based on prime editing outcomes using plasmid or lentiviral expression systems. In this study, we demonstrate that for prime editor (PE) ribonucleoprotein complexes, the auto-inhibitory interaction between the PBS and the spacer sequence affects pegRNA binding efficiency and target recognition. Destabilizing this auto-inhibitory interaction by reducing the complementarity between the PBS-spacer region enhances prime editing efficiency in multiple prime editing formats. In the case of end-protected pegRNAs, a shorter PBS length with a PBS-target strand melting temperature near 37°C is optimal in mammalian cells. Additionally, a transient cold shock treatment of the cells post PE-pegRNA delivery further increases prime editing outcomes for pegRNAs with optimized PBS lengths. Finally, we show that prime editor ribonucleoprotein complexes programmed with pegRNAs designed using these refined parameters efficiently correct disease-related genetic mutations in patient-derived fibroblasts and efficiently install precise edits in primary human T cells and zebrafish.
| S-EPMC10359601 | biostudies-literature
Project description:CRISPR editing involves double-strand breaks in DNA with attending insertions/deletions (indels) that may result in embryonic lethality in mice. The prime editing (PE) platform uses a prime editing guide RNA (pegRNA) and a Cas9 nickase fused to a modified reverse transcriptase to precisely introduce nucleotide substitutions or small indels without the unintended editing associated with DNA double-strand breaks. Recently, engineered pegRNAs (epegRNAs), with a 3'-extension that shields the primer-binding site of the pegRNA from nucleolytic attack, demonstrated superior activity over conventional pegRNAs in cultured cells. Here, we show the inability of three-component CRISPR or conventional PE to incorporate a nonsynonymous substitution in the Capn2 gene, expected to disrupt a phosphorylation site (S50A) in CAPN2. In contrast, an epegRNA with the same protospacer correctly installed the desired edit in two founder mice, as evidenced by robust genotyping assays for the detection of subtle nucleotide substitutions. Long-read sequencing demonstrated sequence fidelity around the edited site as well as top-ranked distal off-target sites. Western blotting and histological analysis of lipopolysaccharide-treated lung tissue revealed a decrease in phosphorylation of CAPN2 and notable alleviation of inflammation, respectively. These results demonstrate the first successful use of an epegRNA for germline transmission in an animal model and provide a solution to targeting essential developmental genes that otherwise may be challenging to edit.
| S-EPMC10939748 | biostudies-literature
Project description:The prime editors (PEs) have shown great promise for precise genome modification. However, their suboptimal efficiencies present a significant technical challenge. Here, by appending a viral exoribonuclease-resistant RNA motif (xrRNA) to the 3'-extended portion of pegRNAs for their increased resistance against degradation, we develop an upgraded PE platform (xrPE) with substantially enhanced editing efficiencies in multiple cell lines. A pan-target average enhancement of up to 3.1-, 4.5- and 2.5-fold in given cell types is observed for base conversions, small deletions, and small insertions, respectively. Additionally, xrPE exhibits comparable edit:indel ratios and similarly minimal off-target editing as the canonical PE3. Of note, parallel comparison of xrPE to the most recently developed epegRNA-based PE system shows their largely equivalent editing performances. Our study establishes a highly adaptable platform of improved PE that shall have broad implications.
| S-EPMC8986804 | biostudies-literature
Project description:Prime editing has gained significant attention as a next-generation gene editing technology, owing to its unique advantages. However, realizing its potential in vivo requires effective delivery strategies. While adeno-associated virus (AAV) has been employed for in vivo delivery of prime editors in research settings, it presents inherent limitations related to vector size, ongoing expression, and inability to re-dose patients. Conversely, lipid nanoparticles (LNPs) do not face these limitations and are emerging as a leading non-viral approach for the delivery of gene editors. In this study, we demonstrate successful co-delivery of chemically modified pegRNA and prime editor mRNA using LNPs for in vivo prime editing. We investigate the impact of pegRNA chemical modifications on editing efficiency and explore different re-dosing regimens. In a daily-repeat dose regimen, we saw striking liver toxicity and no increase in editing; by contrast, weekly-repeat dosing was well tolerated and enabled 1.8-fold increase in editing efficacy. Furthermore, in the NSG immunodeficient mouse model, the efficacy of LNP-delivered prime editing was enhanced by 2.8-fold. In addition, the nature of the ionizable lipids and phospholipids strongly influenced prime editing efficiency in vivo. Overall, these findings will greatly contribute to the future development of LNPs as a robust platform for delivering prime editors in vivo, fostering progress in prime editing research and therapeutic applications.
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| S-EPMC10120949 | biostudies-literature
Project description:Prime editor (PE), which is developed by combining Cas9 nickase and an engineered reverse transcriptase, can mediate all twelve types of base substitutions and small insertions or deletions in living cells but its efficiency remains low. Here, we develop spegRNA by introducing same-sense mutations at proper positions in the reverse-transcription template of pegRNA to increase PE's base-editing efficiency up-to 4,976-fold (on-average 353-fold). We also develop apegRNA by altering the pegRNA secondary structure to increase PE's indel-editing efficiency up-to 10.6-fold (on-average 2.77-fold). The spegRNA and apegRNA can be combined to further enhance editing efficiency. When spegRNA and apegRNA are used in PE3 and PE5 systems, the efficiencies of sPE3, aPE3, sPE5 and aPE5 systems are all enhanced significantly. The strategies developed in this study realize highly efficient prime editing at certain previously uneditable sites.
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