When it was coupled with paired pegRNAs, WT-PE achieved efficient large-scale genomes editing, including large fragment deletion and chromosomal translocation. Different from the traditional prime editor (PE2), this novel system simultaneously introduced a DSB and a 3′ extended flap at the target site, which were then integrated into the genomes by endogenous mechanisms. Here we designed a novel prime editing system by fusing RT with nuclease wild-type Cas9 (WT-PE) to perform large scale genomic manipulation. 10 Although current prime editors are efficiency in introducing small scale DNA editing in a variety of organisms, 11, 12, 13, 14 their efficiency in large scale DNA manipulation remains less efficient. The nick sgRNA introduces a second SSB in the opposite strand to direct DNA repair to use the edited strand as a template, thereby improving the integration of the edits to the genome. Eventually, the flap is integrated to the genome via endogenous DNA repair or replication mechanisms.
Then the 3′ end of DNA in the duplex is extended by RT according to the information encoded by the pegRNA to form a 3′ extended flap. Concerted actions of these components generate an ssDNA break (SSB) in the target site that is recognized by the prime binding site (PBS) of the pegRNA to form an RNA-DNA heteroduplex. 10 Current prime editors are composed of a Cas9 nickase (H840A for spCas9) and an engineered reverse transcriptase (RT) fusion protein (PE2), a prime editing guide RNA (pegRNA), and a nick single guide RNA (sgRNA).
Genome editing technology, especially the recently developed prime editing (PE) technique has enabled targeted introduction of multiple types of edits to the genome, including deletion, insertion, and base substitution, while without requiring the generation of double strand breaks or donor templates. 5, 8, 9 Although the importance of large genomic aberrations in genetic diseases and cancers has been widely recognized, our knowledge about the detailed mechanisms and treatments of these disorders is hurdled in part by lacking an efficient and precise editing tool capable of manipulating the aberrations. 4, 5, 6, 7 These aberrations dramatically alter the content and structure of the involved chromosome, leading to uncontrolled gene function or large-scale dysregulated gene expression. 1, 2, 3 They are also common somatic alterations found in human cancer cells and often function as driving forces for the disease on-set or advance. Therefore, our WT-PE system has great potential to model or treat diseases related to large-fragment aberrations.Īberrations of large chromosomal regions, including duplication, deletion, translocation, and other structural changes, are the cause of a subtype of hereditary genetic disorders, such as thalassemia and hemophilia A.
Coupled with paired prime editing guide RNAs (pegRNAs) that have complementary sequences in their 3′ terminus while target different genomic regions, WT-PE produced bi-directional prime editing, which enabled efficient and versatile large-scale genome editing, including large fragment deletion up to 16.8 megabase (Mb) pairs and chromosomal translocation. WT-PE system simultaneously introduced a double strand break (DSB) and a single 3′ extended flap in the target site. Here, we designed a novel prime editor by fusing reverse transcriptase (RT) to nuclease wild-type Cas9 (WT-PE) to edit large genomic fragment. The current prime editor, PE2, consisting of Cas9-nickase and reverse transcriptase enables efficient editing of genomic deletion and insertion, however, at small scale. Large scale genomic aberrations including duplication, deletion, translocation, and other structural changes are the cause of a subtype of hereditary genetic disorders and contribute to onset or progress of cancer.
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