Webinar: Advances in Improving HDR Efficiency for CRISPR Experiments
Front Line Genomics hosted the webinar Improved Methods for CRISPR Homology-Directed Repair Using Alt-R HDR Enhancer and ssDNA Donors with Optimized Design, which you can watch on demand for free here.
The webinar described how to overcome some of the bottlenecks to using the CRISPR-Cas9 for genome manipulation and ensure its efficacy in a clinical context.
Webinar participants submitted questions to our expert speakers, Mollie Schubert, Research Scientist at Integrated DNA Technologies and Justin McDonough, Scientist at The Jackson Laboratory for Genomic Medicine, which they have answered below.
Can you explain how the HDR Design Tool works?
- The Alt-R™ HDR Design Tool can be used to design HDR donor templates quickly and easily with recommended guides for your target(s). Simply provide basic information about your desired target site, and then use the HDR tool to design and visualize your desired edit within the sequence. The HDR Design Tool will provide the recommended gRNA(s) and HDR donor template for your desired specifications. The tool uses design rules based on extensive wet bench testing including silent mutation incorporation using empirically defined rules to provide the highest HDR rates.
This presentation seemed to focus only on small insertions or point mutations. Have you seen these same performance improvements scale up to larger (multiple kilobase) insertions?
- We have successfully generated insertions up to 1.6 kb using Megamer™ ssDNA fragments with 200 nt homology arms. For larger insertions, plasmid donors with longer (~500 nt) homology arms have been reported to lead to successful HDR. We see that including the Alt-R HDR Enhancer improves HDR rates with both long ssDNA and plasmid DNA donors, similar to smaller mutations or point mutations.
Will this improve HDR efficiency in iPSCs?
- Yes—Justin McDonough, PhD, from the Jackson Laboratories, showed very compelling data on optimized methods for efficient HDR in human iPSCs using Alt-R HDR Enhancer, Alt-R HDR modified donors, and a cold shock treatment after delivery of the CRISPR editing reagents in the second half of this webinar. We recommend reading his recent Methods publication for more detailed information.
When should I consider using a nickase for my HDR instead of the WT Cas9?
- Efficiently generating a break in the target DNA is a critical factor for success in HDR experiments. If there are no efficient Cas9 guides near the intended mutation, then using paired guides with the Cas9 D10A nickase may be a good strategy. For nickase designs, the guides must be oriented with their PAMs facing away from one another in order to target opposite strands to create a double stranded break. We have found that 35-70 nt between the two nick sites is the optimal spacing for robust HDR with Cas9 D10A nickase. This allows for the use of guides further away from the desired mutation and can expand the available options at certain target sites. The Alt-R HDR Design Tool will support nickase designs to help select guides and design donors following our best recommendations.
What should I do if there are no Cas9 guides available where I’d like to do HDR?
- We have developed a novel Cas12a mutant, Alt-R A.s. Cas12a (Cpf1) Ultra, which provides increased levels of genome editing and thus higher HDR rates over the WT version of this enzyme. With the higher activity enabled by using Cas12a Ultra we see similar HDR rates between Cas9 and Cas12a, so both nucleases are great options for HDR experiments, and their use will vary depending on PAM site availability near your desired mutation.
You can catch up on the full webinar here.
© 2019 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.