Many human genomic loci are associated with genetic diseases. Nowadays, it is possible to directly modify the genome to treat genetic diseases. Genetic editing has been achieved on animal models for the purpose of treating human genetic diseases. However, simultaneous induction of DNA double-strand breaks in a large number of cells may result in ectopic and inversion of the genome.
David liu and colleagues fused the catalytically active Cas9 with deaminase, and under the guidance of sgRNA, realized the base substitution of the corresponding sites, and established a single base editing system. C-T, A-G conversion can be performed without DNA double-strand breaks.
However, several laboratories have reported that single-base editing tools can lead to severe off-targeting in the genome and transcriptome range, as well as editing such as insertion and replacement. These problems greatly limit the application of single-base editing tools in the treatment of genetic diseases. . Recently, the David liu team published a paper entitled "Search-and-replace genome editing without double-strand breaks or donor DNA" in Nature, and established a new gene editing system called Prime editing. In the absence of DNA double-strand breaks and without the use of DNA templates, DNA insertion, deletion and any single base substitution can be efficiently produced at yeast and mammalian cell target sites.
They formed a fusion protein between nCas9 (H840A) and M-MLV reverse transcriptase and constructed a new RNA: pegRNA. A process called "PE1" was constructed by realizing the process of efficiently inserting, deleting and single-base replacement of the target site to complete the gene editing process. The reverse transcription amino acid sequence of M-MLV was modified, and another sgRNA opposite to the direction of pegRNA was introduced to construct a PE2 and PE3 system with better thermal stability and the purpose of cutting non-complementary strands respectively. With higher editing efficiency and lower Indels risk, it will greatly promote basic research and clinical gene therapy research in biomedicine.
Compared with the previous gene editing system, the PE system avoids the generation of DNA double-strand breaks, improves the efficiency of gene editing, and expands the scope of application. However, the components of Prime editing are too large to be clinically applied in vivo.Furthermore, the safety of overexpression of reverse transcriptase in cells remains a problem to be considered.