Abstract
<title>Abstract</title> <p> Precise genome editing technologies create the potential for genetic studies and innovative gene therapies. However, the presence of the repair template at the DNA cleavage site could limit the efficiency of homology-directed repair (HDR) editing. On this basis, we explored the possibility of creating an all-in-one RNP cargo containing the guide RNA (gRNA), the Cas9 and the single-stranded oligodeoxynucleotide (ssODN) template. 3D modelling revealed that the ssODN template could interact with the RNP. Here, we present a new CRISPR-Cas9 strategy, called ZIP CRISPR, to reinforce this all-in-one cargo, using complementary extensions of the ssODN template and gRNA, allowing for improved HDR editing efficiency. This new strategy is easy-to-design, easy-to-use, inexpensive and versatile. It increases HDR editing efficiency using Cas9 nuclease up to 12-fold with a mean increase of 5-fold as demonstrated at many <italic>loci</italic> in many cell types. It can also be used with the Cas9 nickase, resulting in HDR editing with minimal InDels, and preventing double-strand break (DSB)-mediated genotoxicity. ZIP CRISPR is a non-viral platform adaptable to targeted DSB (higher HDR editing efficiency) or nick (higher safety) to precisely model and correct a wide range of edits. It is suitable for many biological applications and could be considered for HDR-based gene therapies. </p>