Optimization of Prime Editing System and Its Application in Large DNA Fragment Editing
Gene editing technology utilizes artificial nucleases to insert,replace,or delete specific sequences in desired genomic regions.The discovery of CRISPR/Cas9 nucleases was a milestone in the development of advanced gene editing tools,which revolutionized the field due to their simplicity and versatility.However,the limited precision of Cas9 nucleases remains a notable obstacle.Recently,derivative technologies such as prime editing have earned considerable attention for their enhanced efficiency and precision.The prime editing system consists of two components:the SpCas9 nickase(H840A)fused with reverse transcriptase(MLV-RT)and an engineered prime editing guide RNA(pegRNA).This system can irreversibly introduce various types of genetic changes into the genome,including 12 possible types of point mutations,as well as insertions,deletions and their combinations,without the need for DNA double-strand breaks(DSBs)or donor DNA templates.Prime editing offers several advantages in terms of editing accuracy,versatility,PAM constraints,and off-target effects.The editing results of prime editing system is highly accurate and can be tailored to specific needs.In addition,the system can be edited near or far from PAM sites,making it less constrained by PAM site restrictions.Moreover,it demonstrates high genome-wide specificity.The system also supports a variety of edits,demonstrating immense potential,especially in large DNA fragment editing—an area that relied heavily on CRISPR/Cas9 nucleases before.The development of prime editing,especially bi-direction prime editor,shed new light on large DNA fragment manipulations,including deletions,insertions,replacements,gene integration,as well as chromosomal translocations,inversions,and tandem duplications.Despite the significant progress made with prime editing technology,its application still faces challenges,especially low editing efficiency,which limits its potential in broader research and clinical settings.Consequently,researchers are exploring strategies to enhance the efficiency of prime editing.This review highlights several approaches to improving prime editing efficiency.These include optimizing pegRNA by refining PBS and RT parameters,increasing pegRNA stability and expression levels,and developing automated pegRNA design software.Additionally,efforts are being made to optimize the prime editing system proteins,such as screening for Cas9 and reverse transcriptase variants and performing codon optimization.The final aspect is the regulation of endogenous factors,including the inhibition of mismatch repair mechanisms and the modulation of chromatin environment.These approaches significantly enhance the practicality of prime editing in research and clinical contexts.In conclusion,prime editing represents a major advancement in the field of gene editing,offering powerful tools and methods for both basic research and clinical applications.This review will introduce the discovery,improvement and applications of prime editors,with a focus on prime editing mediated large DNA fragment manipulations.Hopefully,these insights will serve as valuable references for future research and applications of prime editing technology.
CRISPR/Cas9prime editingpegRNAlarge DNA fragment editing
万方数据
维普
