Targeted, programmable, and precise tandem duplication in the mammalian genome.

Tandem duplications are frequent structural variations of the genome and play important roles in genetic disease and cancer. However, interpreting the phenotypic consequences of tandem duplications remains challenging, in part owing to the lack of genetic tools to model such variations. Here, we developed a strategy, tandem duplication via prime editing (TD-PE), to create targeted, programmable, and precise tandem duplication in the mammalian genome.

Erratum: HDAC inhibitors improve CRISPR-Cas9 mediated prime editing and base editing.

[This corrects the article DOI: 10.1016/j.omtn.

HDAC inhibitors improve CRISPR-Cas9 mediated prime editing and base editing.

Recent advances in CRISPR-Cas9 techniques, especially the discovery of base and prime editing, have significantly improved our ability to make precise changes in the genome. We hypothesized that modulating certain endogenous pathway cells could improve the action of those editing tools in mammalian cells. We established a reporter system in which a small fragment was integrated into the genome by prime editing (PE).

Bi-PE: bi-directional priming improves CRISPR/Cas9 prime editing in mammalian cells.

Prime editors consisting of Cas9-nickase and reverse transcriptase enable targeted precise editing of small DNA pieces, including all 12 kinds of base substitutions, insertions and deletions, while without requiring double-strand breaks or donor templates. Current optimized prime editing strategy (PE3) uses two guide RNAs to guide the performance of prime editor. One guide RNA carrying both spacer and templating sequences (pegRNA) guides prime editor to produce ssDNA break and subsequent extension, and the other one produces a nick in the complementary strand.

WT-PE: Prime editing with nuclease wild-type Cas9 enables versatile large-scale genome editing.

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. The current prime editor, PE2, consisting of Cas9-nickase and reverse transcriptase enables efficient editing of genomic deletion and insertion, however, at small scale. Here, we designed a novel prime editor by fusing reverse transcriptase (RT) to nuclease wild-type Cas9 (WT-PE) to edit large genomic fragment.

Expanding PAM recognition and enhancing base editing activity of Cas9 variants with non-PI domain mutations derived from xCas9.

The recognition of protospacer adjacent motif (PAM) is a key factor for the CRISPR (i.e. clustered regularly interspaced short palindromic repeats)/CRISPR-associated 9 (Cas9) system to distinguish foreign DNAs from the host genome, and also significantly restricts the targeting scope of the system during genome-editing applications.

A universal strategy for AAV delivery of base editors to correct genetic point mutations in neonatal PKU mice.

Base editing tools enabled efficient conversion of C:G or A:T base pairs to T:A or G:C, which are especially powerful for targeting monogenic lesions. However, correction of disease-causing mutations is still less efficient because of the large size of base editors. Here, we designed a dual adeno-associated virus (AAV) strategy for delivery of base editors, in which deaminases were linked to Cas9 through the interaction of GCN4 peptide and its single chain variable fragment (scFv) antibody.

Random-PE: an efficient integration of random sequences into mammalian genome by prime editing.

Prime editing (PE) enables efficiently targeted introduction of multiple types of small-sized genetic change without requiring double-strand breaks or donor templates. Here we designed a simple strategy to introduce random DNA sequences into targeted genomic loci by prime editing, which we named random prime editing (Random-PE). In our strategy, the prime editing guide RNA (pegRNA) was engineered to harbor random sequences between the primer binding sequence (PBS) and homologous arm (HA) of the reverse transcriptase templates.

A split cytosine deaminase architecture enables robust inducible base editing.

Directed base substitution with base editing technology enables efficient and programmable conversion of C:G or A:T base pairs to T:A or G:C in the genome. Although this technology has shown great potentials in a variety of basic research, off-target editing is among one of the biggest challenges toward its way to clinical application. Base editing tools, especially the tools converting C to T, caused unpredictable off-target editing throughout the genome, which raise the concern that long-term application of these tools would induce genomic instability or even tumorigenesis.