During lunch time on March 30th, a luncheon seminar hosted by Integrated DNA Technologies, K. K. will be held at the main conference hall. Lunch boxes will be distributed to all participants.

Improved methods for large HDR knock-ins using Alt-R™ HDR Donor Blocks and Alt-R HDR Enhancer V2

Presenter:

Jessica Woodley, Bernice Thommandru, Mollie Schubert, Karthik Murugan, Gavin Kurgan, Matthew McNeill, Ashley Jacobi, Garrett Rettig Integrated DNA Technologies, Inc. Coralville, IA, USA

CRISPR-based homology-directed repair (HDR) is an invaluable tool to facilitate specific mutations in a genomic region of interest in research studies. While many methods have been reported for improving HDR efficiency, achieving precise changes via HDR remains a challenge particularly for large knock-ins.

HDR repair outcomes are most efficient with single-stranded DNA (ssDNA) templates when small insertions, deletions, or SNP changes are desired edits. For these applications, synthetic oligonucleotides (ssODN) have been studied and optimized with modifications for enhanced efficacy in HDR. Larger insertions can be generated via HDR using enzymatically generated ssDNA or double-stranded DNA (dsDNA) donor templates.

Here, we present research demonstrating that improved HDR efficiency for large insertions can be obtained when dsDNA donor templates include novel end-modifications. These modifications have been shown to improve the frequency of HDR and reduce homology-independent (blunt) insertion events that can occur at both on- and off-target CRISPR edits relative to unmodified dsDNA.

In our experiment(s), the use of Alt-R modified dsDNA improved the ratio of HDR:Blunt repair events 4.3-fold on average relative to unmodified dsDNA templates for short inserts and reduced blunt insertion of large templates 4.6-fold at a mock off-target site.

Experimental results demonstrated further improvement to HDR rates when using Alt-R HDR Enhancer V2, a small molecule that increases the rate of HDR in varied cell types including iPSCs and primary human T-cells. Together the use of Alt-R modified repair templates and the Alt-R HDR Enhancer V2 showed improved HDR rates up to 5- to 10-fold across knock-in experiments.

Finally, we present investigations into design considerations for large dsDNA HDR templates including homology arm length and the placement of blocking mutations to disrupt Cas9 re-cleavage.