6 November 2019

Paper of the month september 2019

Search-and-replace genome editing without double-strand breaks or donor DNA

Anzalone AV1,2,3, Randolph PB1,2,3, Davis JR1,2,3, Sousa AA1,2,3, Koblan LW1,2,3, Levy JM1,2,3, Chen PJ1,2,3, Wilson C1,2,3, Newby GA1,2,3, Raguram A1,2,3, Liu DR4,5

One of the most common ways of performing genome editing is to use programmable nucleases such as CRISPR-Cas9 to generate double-strand DNA breaks (DSBs) near the locus of interest, and provide the cells with exogenous donor DNA repair templates so that they can undergo homology-directed repair (HDR) to install precise DNA changes. However, DSB mediated editing will bring in troublesome byproducts of indels and translocations, and the HDR mediated editing is not efficient. Previously, David Liu’s group developed a method called base editing to improve editing efficiency and reduce editing byproducts. However, base editing can only work on very small genomic locus windows.

In this paper, David Liu’s group developed a “search-and-replace” method, called prime editing, which is based on fusing catalytically impaired Cas9 with an engineered reverse transcriptase, to improve editing efficiency and reduce byproduct. Prime editing uses a prime editing guide RNA (pegRNA), which on one hand can lead the enzyme complex to the genomic locus of interest, on the other hand acting as a template for reverse transcription. They also used another gRNA to make a nick on the non-edited strand to favor the maintenance of the reverse transcribed product strand in the genome. By using this method, they achieved ~20-50% desired edit in HEK293T cells with 1-10% indels.

Compare to HDR mediated editing, prime editing gives more desire edits and fewer indels. While comparing to base editing, prime editing is less constrained by the PAM sequence and the editing length. However, more about prime editing needs to be further studied, for example, the off-target effects, the relatively low fidelity of reverse transcriptase, and how to further reduce indel formation.