| Due to the increasing availability of genomic sequences for many species and their usage for basic as well as applied research, a tool that allows specific modifications of a certain gene is needed. The past two decades have brought great progress in developing tools for targeted genome modification [i.e., the zinc-finger nucleases (ZFNs) [1] and transcription activator-like effector nucleases (TALENs)] [2] that are able to cleave double-stranded DNA at a specific region. However, both techniques remain expensive and the generation of proteins capable of binding targeted DNA is a time-consuming process. RNA-guided endonucleases have also been used for genome modification. Using two components of the clustered regularly interspaced short palindromic repeats and CRISPR-associated (CRISPR/ Cas) prokaryote immune system, it was possible to introduce double-stranded breaks in target DNA [3]. In this system, the Cas protein (i.e., Cas9) is guided by short RNA sequence (sgRNA), to the specific region of genome that is cleaved within both DNA strands. This is possible because of the presence of 18–۲۲ nucleotides (called spacer) in sgRNA sequence that are complementary to the targeted DNA. When this break is repaired via the preferential nonhomologous end-joining DNA repair pathway, random insertions or deletions (indels) are introduced in the target sequence(s) [3]. Discovery of novel Cas endonucleases and the development of mutated versions of known Cas endonucleases has increased the specificity and efficiency of these techniques [4]. Another breakthrough in genome editing was the utilization of RNA-guided endonucleases for base editing, including all four transitions: C→T, T→C, A→G, and G→A [5]. A major limitation of the current genome editing technologies has been the ability to provide the altered customized sequence simultaneously at the target site. Recently, a method to overcome such challenges, known as prime editing, has been described by Anzalone et al. in Nature. Prime editing enables the introduction of indels and all 12 base-tobase conversions (both transitions and transversions) without inducing a DNA double-strand break [6]. Here, a prime editing guide RNA (pegRNA) drives the Cas9 endonuclease. Moreover, the pegRNA contains not only the spacer that is complementary to one DNA strand but also a primer binding site (PBS) region and the sequence that will be introduced to the targeted gene. The PBS region is complementary to the second DNA strand and will create a primer for the reverse transcriptase (RT) that is linked to the Cas9(H840A) nickase. The RT is an RNA-dependent DNA polymerase that uses the sequence from the pegRNA as a template. Then the information is copied directly from the pegRNA into target DNA sequence; therefore, altering the preselected target sequence in a customized manner. |
