![]() Parallel lines of research have focused on defining the cellular response to editing reagents with the goal of redirecting repair events through desired repair pathways 14, 15. Gains in nonviral HDR efficiency have been achieved through the optimization of editing reagents, including protein engineering of Cas9 and related nucleases 6, improving the delivery of reagents into cells 7, biophysical optimization of RNP parameters 8, optimization of size and orientation of the homology region of template DNA 9, 10 and tethering template to editing reagents 11, 12, 13. ![]() Strategies to increase HDR frequency may therefore improve outcomes and decrease costs in laboratory and biomedical workflows. The use of HDR to introduce new DNA sequence into targeted locations enables exciting gain-of-function applications 5. These RNPs introduce DSBs at targeted regions in the genome, which are then repaired by error-prone end joining (EJ) processes that rejoin the ends of the break, or homology-directed repair (HDR) processes that resolve DSBs using sequence encoded in a separate template molecule 1 (Extended Data Fig. One high-efficiency nonviral gene editing strategy codelivers ribonucleoprotein (RNP) formulations comprising the targeted nuclease Cas9, a single guide RNA (sgRNA) and a template molecule that contains homology to the region being edited as well as the sequence to be modified or inserted 4. Improved nonviral gene editing would be a powerful approach to unraveling DNA repair mechanisms, a useful laboratory technique and a promising strategy for the treatment of a multitude of diseases 3. The use of nonviral template DNA is thus an appealing alternative, but the efficiency and acute toxicity of nonviral templates can be inferior to viral delivery 2. Although effective, viral workflows are expensive, difficult to scale and potentially toxic to cells. In translational applications, template molecules are often delivered by viral vectors. These latter gene replacement events require the delivery of template DNA encoding new sequences to levels that support gene replacement but do not adversely affect cell viability. Depending on the repair pathway that is engaged, outcomes can include disruption of the targeted gene or replacement with a new sequence that restores or introduces functionality 1. CRISPR–Cas9 enables gene editing via DNA double-strand break (DSB) generation and subsequent activation of cellular DNA repair pathways.
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