Targeted genomic translocations and inversions generated using a paired prime editing strategy

Kweon, Jiyeon; Hwang, Hye‐Yeon; Ryu, Haesun; Jang, An-Hee; Kim, Daesik; Kim, Yongsub

doi:10.1016/j.ymthe.2022.09.008

Cited by 36 publications

(31 citation statements)

References 30 publications

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“…2), using two pegRNAs to simultaneously edit both DNA strands expands the size and type of genomic manipulations possible with prime editing. Many prime editing approaches that use two pegRNAs have been reported, including dual-pegRNA, HOPE (homologous 3′ extension mediated prime editor), PRIME-Del, PEDAR (PE-Cas9-based deletion and repair), twinPE, GRAND (genome editing by RTTs partially aligned to each other but non-homologous to target sequences within duo pegRNA), Bi-PE, bi-WT-PE and PETI (prime editor nuclease-mediated translocation and inversion) [55][56][57]99,[101][102][103][104][105] (Fig. 4a).…”

Section: Prime Editing With Paired Pegrnasmentioning

confidence: 99%

“…However, although PEDAR can make these desired editing outcomes with up to 27% efficiency, they are typically outnumbered by indel byproducts. In independent studies, Yao, Kim, and their respective co-workers developed bi-directional WT-PE (bi-WT-PE) and PETI systems that use a Cas9 nuclease prime editor similar to PEDAR 104,105 (Fig. 4a).…”

Section: Large Sequence Deletions and Insertionsmentioning

confidence: 99%

“…Creating a DSB instead of a DNA nick at the junction of flap synthesis may circumvent these steps and engage other DNA repair pathways to resolve the intermediate towards the intended edit, albeit with the undesired consequence of inducing more indel byproducts from endjoining processes. As discussed above, PEDAR, bi-WT-PE and PETI use this approach to mediate large sequence deletions, inversions and translocations 57,104,105 .…”

Section: Prime Editors With Nucleases That Make Dsbsmentioning

confidence: 99%

“…Fig.4| Prime editing variants. a, Prime editing with paired prime editing guide RNAs (pegRNAs) that template two 3′ DNA flaps containing the edit[55][56][57]99,[101][102][103][104][105] . DNA intermediates formed after prime editor (PE)-mediated nicking and flap extension at the pegRNA target sites are shown on the left.…”

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Prime editing for precise and highly versatile genome manipulation

2022

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single-stranded DNA (ssDNA) at the target site, triggering deamination by the tethered ssDNA-specific deaminase enzyme. Cytosine base editors (CBEs) and adenine base editors (ABEs) contain deaminases that catalyse C•G-to-T•A and A•T-to-G•C changes, respectively [27][28][29][30][31] . C•G-to-G•C base editors (CGBEs) are similar to CBEs, but stimulate replacement of the deaminated cytosine base with guanine, albeit with lower typical efficiencies and product purities compared to CBEs and ABEs [33][34][35][36] . Compared to Cas nucleases, base editors exhibit substantially greater efficiency with few indel byproducts, and show far fewer undesired consequences of DSBs than nucleases in side-by-side comparisons 19,21,[23][24][25][26]37,38 .Because base editors deaminate within a small window of 4-5 nucleotides (nt) canonically, C or A nucleotides very close to the target C or A can also undergo conversion, resulting in 'bystander editing'. The application of base editors to make changes in the coding sequence of genes usually results in only synonymous mutations within a typical base editing activity window 39 owing to the frequency of transition mutations being silent in the genetic code. Nevertheless, undesired base editing of bystander nucleotides can be challenging to avoid in some cases. Base-editing activity is also restricted by the targeting scope of the Cas domain, which requires the presence of a protospacer adjacent motif (PAM) sequence at a specific distance range (typically 15 ± 2 nt) from the target nucleotide. Moreover, some base editors can induce off-target mutations in DNA and RNA [40][41][42] . Although engineering efforts have mitigated many of these drawbacks [43][44][45][46][47][48][49][50][51] , current base editors can only make six of the 12 possible types of point mutation, leaving many other classes of possible DNA edits, such as insertions, deletions and most transversions, beyond the reach of base editing.Motivated by the need for a precise and highly versatile geneediting technology, prime editing enables all types of DNA substitution, small insertions and small deletions to be installed at targeted sites in the genomes of living cells without directly forming DSBs 52 (Fig. 1c). Prime editing minimally requires a prime editor (PE) protein, which is typically a fusion of a nickase Cas9 (nCas9) and a reverse transcriptase (RT), along with a prime editing guide RNA (pegRNA), which specifies the target site for the edit and contains a programmable RNA template for the desired DNA sequence change. To mediate editing, PEs nick the target site on the genome and extend new DNA sequence from this nick using the pegRNA as a template (Fig. 2). This edited DNA strand is then incorporated into the genome through endogenous cellular processes that can be promoted by also nicking the non-edited strand 52 .Through this mechanism of directly rewriting target DNA, prime editing offers extraordinarily high versatility, editing purity and DNA target specificity in comparison to base editing and HDR with nucl...

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Section: Prime Editing With Paired Pegrnasmentioning

confidence: 99%

Section: Large Sequence Deletions and Insertionsmentioning

confidence: 99%

Section: Prime Editors With Nucleases That Make Dsbsmentioning

confidence: 99%

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Prime editing for precise and highly versatile genome manipulation

2022

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“…3b ). This excellent work includes dual-pegRNAs developed in plants 198 , PRIME-Del 199 , HOPE 200 , twin prime editing (twinPE) 201 , GRAND editing 202 , Bi-PE 203 , and PETI 204 , as well as a similar technique PEDAR that fuses Cas9 nuclease to reverse transcriptase (PE-Cas9) and combining it with two pegRNAs 205 . Besides highly efficient replacement and large deletions, twinPE enabled precise large insertions (gene-sized length, >5000 bp) or large inversions (40 kb) in human cells when combined with a site-specific serine recombinase, expanding the capabilities of gene editing for the correction of large or complex human pathogenic alleles 201 , similar with PASTE which uses a CRISPR–Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads (up to 36 kb) 206 .…”

Section: Prime Editorsmentioning

confidence: 99%

Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing

2023

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CRISPR-Cas gene editing has revolutionized experimental molecular biology over the past decade and holds great promise for the treatment of human genetic diseases. Here we review the development of CRISPR-Cas9/Cas12/Cas13 nucleases, DNA base editors, prime editors, and RNA base editors, focusing on the assessment and improvement of their editing precision and safety, pushing the limit of editing specificity and efficiency. We summarize the capabilities and limitations of each CRISPR tool from DNA editing to RNA editing, and highlight the opportunities for future improvements and applications in basic research, as well as the therapeutic and clinical considerations for their use in patients.

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Gene editing therapy for cardiovascular diseases

Wu,

Yang,

Zhang

et al. 2024

MedComm

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The development of gene editing tools has been a significant area of research in the life sciences for nearly 30 years. These tools have been widely utilized in disease detection and mechanism research. In the new century, they have shown potential in addressing various scientific challenges and saving lives through gene editing therapies, particularly in combating cardiovascular disease (CVD). The rapid advancement of gene editing therapies has provided optimism for CVD patients. The progress of gene editing therapy for CVDs is a comprehensive reflection of the practical implementation of gene editing technology in both clinical and basic research settings, as well as the steady advancement of research and treatment of CVDs. This article provides an overview of the commonly utilized DNA‐targeted gene editing tools developed thus far, with a specific focus on the application of these tools, particularly the clustered regularly interspaced short palindromic repeat/CRISPR‐associated genes (Cas) (CRISPR/Cas) system, in CVD gene editing therapy. It also delves into the challenges and limitations of current gene editing therapies, while summarizing ongoing research and clinical trials related to CVD. The aim is to facilitate further exploration by relevant researchers by summarizing the successful applications of gene editing tools in the field of CVD.

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Targeted genomic translocations and inversions generated using a paired prime editing strategy

Cited by 36 publications

References 30 publications

Prime editing for precise and highly versatile genome manipulation

Prime editing for precise and highly versatile genome manipulation

Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing

Gene editing therapy for cardiovascular diseases

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