CRISPR genome editing depends on a Cas enzyme, often Cas9, which cuts genomic DNA in cells. Cells then repair the cut DNA. The two major repair pathways are as follows:
- Non-homologous end joining (NHEJ)—In NHEJ, which is the predominant pathway, broken ends of the DNA are efficiently joined. However, insertions and/or deletions often occur at the breakpoint, leading to gene knockout.
- Homology-directed repair (HDR)—In HDR, cells copy a DNA template to repair the DNA break via homologous recombination.
Taking advantage of the HDR pathway, researchers can add a template carrying desired changes. However, the rates of HDR can be inconsistent or low. IDT has developed a number of products to help improve HDR rates, including Alt-R HDR Donor Oligos, Megamer™ ssDNA Fragments, and Alt‑R HDR Enhancer. Designing the HDR template itself can be challenging, but IDT has developed a web-based, user-friendly tool to make this much easier. In this webinar, Dr Matt McNeill, Bioinformatics Sr Staff Scientist at IDT, describes the reagents and design rules used to improve HDR rates, and he demonstrates how to use the Alt-R HDR Design Tool.
Alt-R donor oligos are ssDNA, and they carry chemical modifications at both ends. The Alt-R HDR Enhancer is a small-molecule compound that inhibits NHEJ, tilting the balance of cell repair pathways in favor of HDR. In the webinar, Dr McNeill describes experiments demonstrating the benefits of including Alt-R donor modifications and Alt-R HDR Enhancer in HDR experiments. Data from EcoRI enzymatic cleavage assays, next generation sequencing, and BFP to GFP assays concur, showing that these approaches all improve HDR rates.
Dr McNeill follows up on previous work by explaining how a high-quality gRNA sequence can be selected, and how scoring of on-target and off-target cleavage can impact experimental results. He also explains how to choose optimal homology arm lengths for the donor sequences and how to position the insert relative to the cut site. He goes on to describe how placing silent single nucleotide polymorphisms (SNPs) into HDR donors can prevent Cas9 re-cleavage and improve HDR rates. Finally, he discusses when nickases should be used and how to pick dual gRNAs for a nickase genome editing experiment. Dr McNeill ends the webinar with a demonstration of how to use the Alt-R HDR Design Tool to choose gRNAs and design HDR templates.
Watch the recorded webinar below to learn how to use the Alt-R HDR Design Tool, Alt-R HDR Donor Oligos, and Alt-R HDR Enhancer to achieve high HDR rates in CRISPR experiments.