Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure

Li, Xiaosa; Zhou, Lina; Gao, Bao‐Qing; Li, Guangye; Wang, Xiao; Wang, Ying; Wei, Jia; Han, Weihong; Wang, Zixian; Li, Jifang; Gao, Runze; Zhu, Jing; Xu, Wenchao; Wu, Jing; Yang, Bei; Sun, Xiaodong; Yang, Li; Chen, Jia

doi:10.1038/s41467-022-29339-9

Cited by 75 publications

(62 citation statements)

References 29 publications

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“…Alternatively, expressing pegRNAs with RNA polymerase II also removes the need to avoid poly(U) termination signals 79 . In addition, Chen and co-workers showed higher prime editing efficiency with apegRNAs that stabilize the secondary structure of the second stem-loop within the pegRNA scaffold 80 (Table 1). However, other pegRNA scaffold variants with even lower folding energies did not improve prime editing 80 , suggesting that the sequence determinants of pegRNA efficiency are complex.…”

Section: Table 1 (Continued) | Prime Editor and Pegrna Architecturesmentioning

confidence: 99%

“…In addition, Chen and co-workers showed higher prime editing efficiency with apegRNAs that stabilize the secondary structure of the second stem-loop within the pegRNA scaffold 80 (Table 1). However, other pegRNA scaffold variants with even lower folding energies did not improve prime editing 80 , suggesting that the sequence determinants of pegRNA efficiency are complex. Additional exploration of pegRNA scaffold variants may therefore provide an opportunity for further prime editing improvement.…”

Section: Table 1 (Continued) | Prime Editor and Pegrna Architecturesmentioning

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: Table 1 (Continued) | Prime Editor and Pegrna Architecturesmentioning

confidence: 99%

Section: Table 1 (Continued) | Prime Editor and Pegrna Architecturesmentioning

confidence: 99%

Prime editing for precise and highly versatile genome manipulation

2022

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“…Similarly, Li et al stabilized pegRNA by inserting a C/G pair or changing each non-C/G pair to a C/G pair in the small hairpin of pegRNA(apegRNA), resulting in an average 2.77-fold improvement in indel-editing efficiency when applied it to PE3 [ 31 ]. They also adopted the second strategy: introducing same-sense mutations at appropriate positions in the RT template portion of pegRNA (spegRNA), which increased the base-editing efficiency by an average of 353-fold.…”

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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“…However, a number of modifications proposed further make it possible to increase its efficiency many times over. Optimization of nuclear localization sequence of the genome editor (PE2*) [ 94 ], additional single-strand DNA break (PE3) [ 95 ], modification of gRNA backbone [ 96 ] and blocking DNA mismatch repair mechanisms using transient expression of dominant negative MMR protein (MLH1dn)—PE5 [ 97 ]—all increased the modification efficiency of prime editing up to 15–99% (depending on the edited gene, length and nature of the modification) and reduced the probability of indels to 1–10% [ 96 , 97 ].…”

Section: Genome Editingmentioning

confidence: 99%

“…The latest modifications of prime editing (twinPE) make it possible to overcome this limit and integrate up to 40,000 base pairs into the genome or delete protensive DNA fragments (at least, 800 base pairs long) with an efficiency of up to 80% [ 98 ]. In most cases, such high efficiency of editing systems (including prime editing) has been demonstrated on transformed cell cultures: HEK293, HeLa, K562, and U2OS [ 93 , 94 , 95 , 96 , 97 , 98 ], whereas the efficiency of most genome editors in primary cells and iPSCs remains rather modest. The effectiveness of the latest modifications of prime editing in iPSCs and primary cell cultures has yet to be established, but preliminary studies demonstrate the high potential and flexibility of this editing system.…”

Section: Genome Editingmentioning

confidence: 99%

The Power of Gene Technologies: 1001 Ways to Create a Cell Model

Karagyaur

Primak

Efimenko

et al. 2022

Cells

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Modern society faces many biomedical challenges that require urgent solutions. Two of the most important include the elucidation of mechanisms of socially significant diseases and the development of prospective drug treatments for these diseases. Experimental cell models are a convenient tool for addressing many of these problems. The power of cell models is further enhanced when combined with gene technologies, which allows the examination of even more subtle changes within the structure of the genome and permits testing of proteins in a native environment. The list and possibilities of these recently emerging technologies are truly colossal, which requires a rethink of a number of approaches for obtaining experimental cell models. In this review, we analyze the possibilities and limitations of promising gene technologies for obtaining cell models, and also give recommendations on the development and creation of relevant models. In our opinion, this review will be useful for novice cell biologists, as it provides some reference points in the rapidly growing universe of gene and cell technologies.

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Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure

Cited by 75 publications

References 29 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

The Power of Gene Technologies: 1001 Ways to Create a Cell Model

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