摘要
—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.
摘要译文
- 自从应用CRISPR/CAS技术编辑各种生物的基因组以来已经有10年了。它有能力在研究人员指定的DNA区域中产生双链断裂的能力开始了生物工程革命。后来，开发了基础编辑（BE）方法。BE是通过与脱氨酶和其他酶融合的CAS核酸酶突变形式（Nickase）突变形式通过形成单链断裂的。它可用于促进A \（\ leftrightArrow \）g和c \（\ leftrightArrow \）t跃迁，以及c→G transertersion。就在3年前，发明了CRISPR/CAS的新的Prime编辑（PE）变体。与BE不同，在PE中，Nickase与逆转录酶融合，能够使用Pegrna模板构建新的DNA链。Pegrna由一个细长的导向RNA组成，在3'末端具有额外的序列。主要的编辑使得将所需的突变插入此额外的序列，并在不使用特殊供体DNA的情况下执行碱基的任何替代和插入。迄今为止，已经提出了许多PE变体。在本综述中简要考虑它们，重点是植物基因组的主要编辑。对Pegrna设计计划以及编辑效率的评估也引起了一些关注。如此多种PE技术是由于高精度引入所需变化的机会，而各种生物体的基因组中的脱靶突变频率相当低。主要编辑的效率相对较低，激发了研究人员提供新方法。希望该技术的进一步发展将足以改善PE，以在适合任何生物体的基因组靶向方法中占据应有的地位，并将对农业部门，工业生物技术和医学产生积极影响。
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