CRISPR-Cas proteins introduce double-stranded breaks (DSBs) at targeted genomic loci. These are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Researchers often want to introduce a precise mutation, which requires utilizing the HDR pathway. Providing a ssDNA template during repair allows introduction of a precise desired mutation, but the rates of successful HDR are often low compared to NHEJ-mediated repair.
Methods to improve HDR rates
In the first part of this webinar, Mollie Schubert, MS, IDT Research Scientist, describes methods to improve the rate of HDR vs. NHEJ-mediated repair in various cell lines. The topics include tips for:
- Selection of the right gRNA and CRISPR enzyme
- HDR template design
- Use of Alt-R HDR Enhancer, a small molecule compound that increases the rate of HDR by inhibiting NHEJ-mediated repair
Mollie also describes the upcoming release of the Alt-R CRISPR HDR Design Tool, a novel bioinformatics tool for ssDNA HDR template design. The tool will support designs up to 3 kb, single- and dual-guide designs (i.e., for use with Cas9 nickases), and more.
Improving homology-directed repair efficiency in human stem cells
In the second part of this webinar, Justin McDonough, PhD, Scientist at The Jackson Laboratory for Genomic Medicine, describes methods he and his colleagues have used to improve HDR efficiency in human induced pluripotent stem cells (iPSCs). The topics include:
- Introducing CRISPR-Cas9 components into iPSCs
- Fluorescence and flow cytometry assays for determining the HDR-edited status of iPSCs
- Cell culture parameters to increase HDR rates in iPSCs
- Strategic design considerations for donor oligos to introduce a desired single nucleotide variant in iPSCs
By combining all these optimized methods, The Jackson Laboratory scientists dramatically increased the ratio of HDR to NHEJ when either Cas9 or Cas12a Ultra was used along with the Alt-R HDR Enhancer in iPSCs.
To learn more, view the webinar.