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). Providing a ssDNA template during repair allows researchers to introduce a desired mutation precisely, by utilizing the HDR pathway. However, rates of HDR are often low compared to NHEJ-mediated repair.
Here, we describe methods to improve the rate of HDR vs NHEJ-mediated repair by careful selection of template characteristics, including homology arm lengths, placement of synonymous SNPs in the HDR template, and the addition of chemical modifications to increase DNA stability against cellular nucleases. In addition, we demonstrate improved HDR rates when using Alt-R HDR Enhancer, a small molecule compound that increases the rate of HDR by inhibiting NHEJ-mediated repair. Increased HDR rates have been achieved in various cell lines, including iPSCs.
Furthermore, we implement our findings in the Alt-R CRISPR HDR Design Tool, a novel bioinformatics tool for ssDNA HDR template design. The Alt-R CRISPR HDR Design Tool supports single-stranded designs up to 3 kb, single- and dual-guide designs (i.e., for use with Cas9-Nickases), guide selection suggestions, insertion of synonymous SNPs in coding regions, and more.
When: Thursday, September 5th, 2019
Time: 10am EDT / 3pm BST / 4pm CEST
The webinar will be available on-demand after this date.
Benefits of Attending:
- Learn how to improve HDR rates when using CRISPR genome editing
- Guidance on best practices and design recommendations
- Optimized design of ssDNA donors:
- Selecting the right gRNA and CRISPR enzyme
- Homology arm (HA) length for small vs large insertions
- Silent mutations to prevent re-cutting
- HDR donor modifications – addition of chemical modifications to stabilize donors
- Alt-R HDR Enhancer – a small molecule solution that consistently increases HDR rates
- Alt-R HDR Design tool – a novel bioinformatics tool for ssDNA HDR template design
Mollie Schubert is a Research Scientist in the Molecular Genetics Research Group at Integrated DNA Technologies. Mollie received her master’s degree in biochemistry from Iowa State University, and has been with IDT since 2013. For the past five years Mollie’s studies have focused on CRISPR gene editing. Her research has included high-throughput screening of CRISPR-Cas9 guides for the development of a site selection tool, optimization of composition and delivery for synthetic RNA reagents complexed to recombinant CRISPR nucleases, and the development of methods for efficient gene editing, with emphasis on homology directed repair.
Justin McDonough is a Scientist in the Cellular Engineering group at the Jackson Laboratory. Justin received his Ph.D. in Microbiology and Immunology from the University of North Carolina at Chapel Hill. During postdoctoral work at Yale University, he used high-throughput screening and CRISPR gene editing to probe human gene function. Currently, Justin manages the Genome Editing core service at the Jackson Laboratory which is dedicated to providing precisely edited human induced pluripotent stem cells to the research community. To achieve this goal, his group has focused on optimizing methods for efficient delivery of CRISPR reagents and improved homology directed repair outcomes.