Advances in Improving HDR Efficiency for CRISPR Experiments
Despite vast progress in leveraging the CRISPR-Cas9 system for genome manipulation, bottlenecks still remain. Overcoming these bottlenecks is critical to ensure the efficacy of this technology in a clinical context. Several engineering approaches involve inducing a double -stranded break (DSB) to introduce new mutations, which are then repaired by the endogenous cellular machinery. The two predominant repair pathways are non-homologous end joining (NHEJ) and homology-directed repair (HDR). Although HDR occurs less frequently in cells, it enables the introduction of a specific donor sequence, allowing precise genomic alterations to be made.
Various design principles have been tried and tested to increase HDR efficiency and thereby overcome this rate-limiting step. Recent evidence suggests that single-stranded oligo deoxynucleotide (ssODN) donor templates result in higher insertion efficiency than dsDNA templates. Interestingly, HDR rates also vary depending on the distance between the protospacer adjacent motif (PAM) site and intended insertion site, so optimal crRNA protospacers should be carefully selected.
In our latest webinar, our expert speakers will be sharing their extensive research developing both in vitro and in silico approaches to increase HDR efficiency. Namely, the “Alt-R CRISPR HDR Design Tool”, a novel bioinformatics tool for ssDNA HDR template design. This tool provides robust design recommendations to effectively favour repair by the HDR pathway efficiency, while reducing off-target activity without compromising cell viability.
Want to get the best tips to design and run your own CRISPR experiments? Register for our upcoming webinar this Thursday.