A flexible split prime editor using truncated reverse transcriptase improves dual-AAV delivery in mouse liver

Zheng, Changcheng; Liang, Shun-Qing; Liu, Bin; Liu, Pengpeng; Kwan, Suet‐Yan; Wolfe, Scot A.; Xue, Wen

doi:10.1016/j.ymthe.2022.01.005

Cited by 68 publications

(37 citation statements)

References 28 publications

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“…In addition to exchanging the Cas9 domain, PE variants that utilize alternative RTs have also been established. Multiple groups have observed that the RNaseH domain of the engineered MMLV RT in PE2 is dispensable and can be removed without affecting editing levels 72,86,88,89 .…”

Section: Prime Editor Variantsmentioning

confidence: 99%

“…In many cultured mammalian cell lines, transient lipid-mediated transfection or electroporation of prime editor plasmids can mediate high editing efficiencies, and selection for transfected cells can further improve editing performance 52,53,91 . Hydrodynamic tail-vein injection of plasmid DNA can also deliver PEs into mouse hepatocytes in vivo 57,68,81,88,108 . These approaches induce transient expression of prime editing agents in mammalian cells and thereby limit undesired editing at off-target loci.…”

Section: Dna Transfection and Viral Deliverymentioning

confidence: 99%

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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 Editor Variantsmentioning

confidence: 99%

Section: Dna Transfection and Viral Deliverymentioning

confidence: 99%

Prime editing for precise and highly versatile genome manipulation

2022

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“…In addition, the choice of split sites for the split-PE system is limited. To overcome this limitation, Zheng et al used truncated RT to further reduce the size of PE and improve intracellular safety [ 50 ]. They developed a compact PE that did not contain the RNase H domain of RT, increasing flexibility in the selection of split sites, while maintaining editing efficiency.…”

Section: Improvements In Prime Editorsmentioning

confidence: 99%

Prime Editing: An All-Rounder for Genome Editing

Kuang

Shao

et al. 2022

IJMS

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Prime editing (PE), as a “search-and-replace” genome editing technology, has shown the attractive potential of versatile genome editing ability, which is, in principle, currently superior to other well-established genome-editing technologies in the all-in-one operation scope. However, essential technological solutions of PE technology, such as the improvement of genome editing efficiency, the inhibition of potential off-targets and intended edits accounting for unexpected side-effects, and the development of effective delivery systems, are necessary to broaden its application. Since the advent of PE, many optimizations have been performed on PE systems to improve their performance, resulting in bright prospects for application in many fields. This review briefly discusses the development of PE technology, including its functional principle, noteworthy barriers restraining its application, current efforts in technical optimization, and its application directions and potential risks. This review may provide a concise and informative insight into the burgeoning field of PE, highlight the exciting prospects for this powerful tool, and provide clues for questions that may propel the field forward.

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“…As base editing, prime editing has not yet entered clinical trials due to its immature development. However, the potential of PE has so far been demonstrated during the past 2 years in vitro ( Surun et al, 2020 ; Habib et al, 2022 ; Happi Mbakam et al, 2022 ; Petri et al, 2022 ; Tremblay et al, 2022 ) and in animal models ( Liu et al, 2021b ; Jang et al, 2022 ; Zheng et al, 2022 ; Zhi et al, 2022 ). Unfortunately, the gene modification efficiency is very low in some models.…”

Section: Nickase-based Genome Engineering Technologiesmentioning

confidence: 99%

Improvements of nuclease and nickase gene modification techniques for the treatment of genetic diseases

Mbakam²,

Song³

et al. 2022

Front. Genome Ed.

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Advancements in genome editing make possible to exploit the functions of enzymes for efficient DNA modifications with tremendous potential to treat human genetic diseases. Several nuclease genome editing strategies including Meganucleases (MNs), Zinc Finger Nucleases (ZFNs), Transcription Activator-like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins (CRISPR-Cas) have been developed for the correction of genetic mutations. CRISPR-Cas has further been engineered to create nickase genome editing tools including Base editors and Prime editors with much precision and efficacy. In this review, we summarized recent improvements in nuclease and nickase genome editing approaches for the treatment of genetic diseases. We also highlighted some limitations for the translation of these approaches into clinical applications.

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A flexible split prime editor using truncated reverse transcriptase improves dual-AAV delivery in mouse liver

Cited by 68 publications

References 28 publications

Prime editing for precise and highly versatile genome manipulation

Prime editing for precise and highly versatile genome manipulation

Prime Editing: An All-Rounder for Genome Editing

Improvements of nuclease and nickase gene modification techniques for the treatment of genetic diseases

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