14 November 2019

November 2019

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

Andrew V. Anzalone, Peyton B. Randolph, Jessie R. Davis, Alexander A. Sousa, Luke W. Koblan, Jonathan M. Levy, Peter J. Chen, Christopher Wilson, Gregory A. Newby, Aditya Raguram & David R. Liu

Gene therapy is an approach to treat or cure patients suffering from a genetic disease. Programmable nucleases are an effective tool that can be used for gene therapy. For example, introducing a DNA double-strand break using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Protein 9 (Cas9) results in desired insertions and deletions; however, it also causes undesired outcomes, including complex mixtures of products, translocation or large deletions. Moreover, most genetic diseases need more precise editing, which can be achieved by employing the homology-directed repair (HDR) pathway. HDR that is applied to gene therapy requires an externally-delivered repair template containing the correction mutation. However, this precise way of DNA repair is very inefficient, and therefore, difficult to apply in most of the therapeutically-relevant cells.

To address this issue, the authors developed a new genome-editing technology called “prime editing”, which is as precise as HDR, does not require DNA double strand breaks and hence, does not result in unwanted by-products such as insertion or deletion.

The Prime Editor (PE) consists of a Reverse Transcriptase linked to a Cas9 nickase. In the Cas9 nickase, one of the two nuclease domains is inactivated, and consequently, a nick is introduced in one of the DNA strands instead of a DNA double strand break. The Reverse Transcriptase was originally discovered from retroviruses, where it has been used to replicate the viral genome. The Reverse Transcriptase uses RNA as a template to generate complementary DNA (cDNA), resulting in RNA-DNA hybrids, which will be integrated into the host’s genome and converted to double-stranded DNA by the cell’s DNA replication machinery. The Reverse Transcriptase is widely used in laboratories to generate PCR templates from RNA, since only DNA can serve as a PCR template. Furthermore, the authors of the paper engineered the guide RNA by adding extra nucleotides towards the 3’ end of the trans-activation CRISP RNA (tracrRNA). The additional nucleotides, which are complementary to the Cas9-targeted site, serve as primers and a template for the Reverse Transcriptase. The template contains the desired edits. The authors referred to the sgRNA that was used for prime editing as “pegRNA”.

Throughout several experiments, the authors developed a more efficient version of PE1 by mutating the Reverse Transcriptase, thus optimizing the pegRNA and introducing a second nick in the non-edited strand.

The authors compared prime editing to base editing and concluded that prime editing is the method of choice particularly when bystander mutations cannot be tolerated. Moreover, they showed that prime editing results in much lower off-targets as compared to Cas9 for the tested sgRNAs. Using Cas9 and HDR in contrast to prime editing showed that prime editing resulted in edits with comparable or even higher efficiencies and lower indel rates. They could not detect any toxicity of the Reverse Transcriptase in human cells. Finally, they successfully applied prime editing to correct mutations that cause genetic disease in sickle cell disease, Tay-Sachs disease and prion disease.

The authors concluded that prime editing expands the scope of genome editing tools. However, further research would need to be carried out to fully understand and improve prime editing.