Refined Homology-Directed Repair Methodological Approaches of Donorguide, a Chimeric RNA: DNA tracrRNA

The use of single-stranded oligodeoxynucleotide (ssODN) along with CRISPR-Cas9-mediated double-strand breaks (DSB) is one of the most commonly deployed methods for introducing genetic alterations, but this approach has notable limitations. Recognizing this, we have developed a protocol article that provides a step-by-step process of donorguide, a covalent fusion of trans-activating CRISPR RNA (tracrRNA) and ssODN. Donorguide has the potential to enhance the introduction of specific genetic alterations (insertion, deletion, and substitution) at a DSB, improving homology-directed repair methods from zebrafish in vivo to human cells in vitro. We also explored and discuss the impact of increasing the length of donorguide homology arms in zebrafish.