A new genome editing strategy called prime editing uses a catalytically impaired Cas9 fused to an engineered reverse transcriptase to write desired genetic sequence information directly into a target locus. This ‘search-and-replace’ approach was able to perform targeted insertions, deletions and all possible base-to-base conversions in human cells.

targeted insertions, deletions and all possible base-to-base conversions in human cells

CRISPR–Cas9 and other programmable nucleases can be used for targeted mutagenesis as they generate site-specific DNA double-strand breaks (DSBs), which induces homology-directed repair (HDR). However, HDR is dependent on a DNA donor template and is inefficient in most cell types, and DSBs can induce undesirable insertions and deletions (indels).

Prime editors comprise a reverse transcriptase fused to an RNA-guided DNA-nicking domain, such as Cas9 nickase, which introduces DNA single-strand cuts rather than DSBs. This fusion protein forms a complex with a prime editing extended guide RNA (pegRNA), which both specifies the target site and encodes the desired edit. Once bound to target DNA, the complex nicks the strand containing the protospacer adjacent motif (PAM). This process generates a free 3′ end that hybridizes to a primer-binding site on the pegRNA. Reverse transcription of new DNA including a desired edit then occurs from the template within the pegRNA extension. Preferential excision of the unedited 5′ single-stranded ‘flap’ and ligation of the edited 3′ flap creates a heteroduplex with one edited and one unedited strand that is subsequently resolved by DNA mismatch repair to stably ‘install’ the edit.

Anzalone et al. describe multiple prime editor variants optimized to increase editing efficiencies and minimize indel by-products. Applying prime editing to human HEK293T cells, the team corrected the most common mutation that causes Tay–Sachs disease by removing 4 bp in the gene HEXA. The team also used prime editing to generate and subsequently correct the major cause of sickle cell disease, which is a transversion point mutation in HBB. Moreover, prime editing was used to install a transversion in PRNP that confers resistance to prion disease.

Comparing prime editing to Cas9-initiated HDR, prime editors exhibited higher or similar efficiency with much lower indel formation and off-target activity. Similar to base editors, prime editors circumvent the need for DSBs and donor DNA templates; however, prime editors offer substantial advantages over current base editors when target bases are not well-positioned for editing or when multiple cytosines or adenines are present.