E. V. Mikhaylova, B. R. Kuluev, G. A. Gerashchenkov, D. A. Chemeris, R. R. Garafutdinov, A. R. Kuluev, An. K. Baymiev, Al. K. Baymiev, A. V. Chemeris
{"title":"Prime-Editing Methods and pegRNA Design Programs","authors":"E. V. Mikhaylova, B. R. Kuluev, G. A. Gerashchenkov, D. A. Chemeris, R. R. Garafutdinov, A. R. Kuluev, An. K. Baymiev, Al. K. Baymiev, A. V. Chemeris","doi":"10.1134/s0026893324010084","DOIUrl":null,"url":null,"abstract":"<p><b>Abstract</b>—It has been 10 years since CRISPR/Cas technology was applied to edit the genomes of various organisms. Its ability to produce a double-strand break in a DNA region specified by the researcher started a revolution in bioengineering. Later, the Base Editing (BE) method was developed. BE is performed via the formation of single-strand breaks by the mutant form of Cas nuclease (nickase), fused with deaminases and other enzymes. It can be used to promote A <span>\\( \\leftrightarrow \\)</span> G and C <span>\\( \\leftrightarrow \\)</span> T transitions, and a C → G transversion. Just over 3 years ago, a new Prime Editing (PE) variant of CRISPR/Cas was invented. Unlike BE, in PE the nickase is fused with reverse transcriptase, capable of building a new DNA chain using the pegRNA template. The pegRNA consists of an elongated guide RNA with an extra sequence at the 3'-end. Prime editing makes it possible to insert the desired mutations into this extra sequence and to carry out any substitutions and indels of bases without the use of special donor DNA. To date, a number of PE variants have been proposed; they are briefly considered in this review with an emphasis on prime editing of plant genomes. Some attention is also paid to pegRNA design programs, as well as evaluation of the efficiency of the editing. Such a variety of PE techniques is due to the opportunities of high-precision introduction of desired changes with a rather low frequency of off-target mutations in the genomes of various organisms. The relatively low efficiency of prime editing inspires researchers to offer new approaches. There is hope that further development of the technology will improve PE enough to take its rightful place among the genome targeting methods that are suitable for any organisms, and will have a positive impact on the agricultural sector, industrial biotechnologies, and medicine.</p>","PeriodicalId":18734,"journal":{"name":"Molecular Biology","volume":null,"pages":null},"PeriodicalIF":1.5000,"publicationDate":"2024-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular Biology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1134/s0026893324010084","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract—It has been 10 years since CRISPR/Cas technology was applied to edit the genomes of various organisms. Its ability to produce a double-strand break in a DNA region specified by the researcher started a revolution in bioengineering. Later, the Base Editing (BE) method was developed. BE is performed via the formation of single-strand breaks by the mutant form of Cas nuclease (nickase), fused with deaminases and other enzymes. It can be used to promote A \( \leftrightarrow \) G and C \( \leftrightarrow \) T transitions, and a C → G transversion. Just over 3 years ago, a new Prime Editing (PE) variant of CRISPR/Cas was invented. Unlike BE, in PE the nickase is fused with reverse transcriptase, capable of building a new DNA chain using the pegRNA template. The pegRNA consists of an elongated guide RNA with an extra sequence at the 3'-end. Prime editing makes it possible to insert the desired mutations into this extra sequence and to carry out any substitutions and indels of bases without the use of special donor DNA. To date, a number of PE variants have been proposed; they are briefly considered in this review with an emphasis on prime editing of plant genomes. Some attention is also paid to pegRNA design programs, as well as evaluation of the efficiency of the editing. Such a variety of PE techniques is due to the opportunities of high-precision introduction of desired changes with a rather low frequency of off-target mutations in the genomes of various organisms. The relatively low efficiency of prime editing inspires researchers to offer new approaches. There is hope that further development of the technology will improve PE enough to take its rightful place among the genome targeting methods that are suitable for any organisms, and will have a positive impact on the agricultural sector, industrial biotechnologies, and medicine.
摘要-CRISPR/Cas 技术应用于编辑各种生物的基因组已有 10 年之久。它能在研究人员指定的 DNA 区域产生双链断裂，掀起了一场生物工程革命。后来，碱基编辑（BE）方法被开发出来。碱基编辑是通过突变形式的 Cas 核酸酶（缺口酶）与脱氨酶和其他酶融合形成单链断裂来实现的。它可以用来促进 A \( \leftrightarrow \) G 和 C \( \leftrightarrow \) T 转换，以及 C → G 转换。就在 3 年多前，CRISPR/Cas 发明了一种新的主编辑（PE）变体。与 BE 不同，PE 中的缺口酶与反转录酶融合在一起，能够利用 pegRNA 模板构建新的 DNA 链。pegRNA 由一个拉长的引导 RNA 组成，3'端有一个额外的序列。通过质粒编辑，可以将所需的突变插入到这一额外序列中，并在不使用特殊供体 DNA 的情况下实现碱基的替换和嵌套。迄今为止，已经提出了许多 PE 变体；本综述将简要介绍这些变体，重点是植物基因组的基质编辑。本综述还关注了 pegRNA 设计程序以及编辑效率评估。PE技术之所以种类繁多，是因为在各种生物的基因组中，有机会高精度地引入所需的变化，而脱靶突变的频率却相当低。素材编辑的效率相对较低，这激励着研究人员提供新的方法。希望该技术的进一步发展能改善质粒编辑技术，使其在适用于任何生物的基因组靶向方法中占据应有的地位，并对农业部门、工业生物技术和医学产生积极影响。
期刊介绍：
Molecular Biology is an international peer reviewed journal that covers a wide scope of problems in molecular, cell and computational biology including genomics, proteomics, bioinformatics, molecular virology and immunology, molecular development biology, molecular evolution and related areals. Molecular Biology publishes reviews, experimental and theoretical works. Every year, the journal publishes special issues devoted to most rapidly developing branches of physical-chemical biology and to the most outstanding scientists.
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