HDR: What is it and how can yours improve?

The takeaway: CRISPR-Cas9 and homology directed repair provide a streamlined, powerful way to consistently generate successful knock-ins that can be used in many applications. It is important to test different HDR experimental conditions and select the best ones. This blog walks you through the steps.

What is HDR?

The cell genome is continuously damaged by both external environmental factors such as irradiation or ultraviolet light (UV) and by the cell's own metabolites including nucleases and reactive oxygen species. In the most serious cases this damage can result in double-strand breaks (DSBs) in the chromosome which need to be repaired to avoid cell death.

DSBs can be repaired by one of the following mechanisms: microhomology-mediated end joining (MMEJ), non-homologous end joining (NHEJ), or homology-directed repair (HDR), the latter being the focus of this post.

Briefly, HDR is the process of copying a donor strand of DNA to the place where a DSB occurred. If the homologous DNA sequence that is being used as a template is identical to the original DNA sequence, then no errors should occur as a result of the repair process.

This mechanism can be used for the actual purpose of repair as its name suggests. However, scientists can also leverage this pathway to introduce mutations. In the pre-CRISPR times (before 2009), researchers were introducing DNA templates into cells via viral or plasmid vectors and as linear DNA fragments to encourage cells to incorporate those sequences into their genomes. They noticed that homologous recombination (HR) did occur in cells without DBS but that the presence of DBS made HR much more effective. This key observation has eventually led to the discovery of CRISPR which introduces targeted and precise DBS giving the opportunity for HDR to occur.

Genome engineering via CRISPR and HDR

CRISPR is a great example of disruptive science because it has completely changed the genome engineering landscape and how we think about and approach genome editing experiments. Essentially, all we need right now for a successful knock-in is a Cas enzyme, a guide RNA, and a donor DNA with a targeted sequence of interest. CRISPR-Cas9 coupled with HDR provide an elegant and streamlined solution to consistently generate successful knock-ins that can be used in many applications.

Tips for a successful knock-in experiment.

Some of the recommendations for a successful HDR experiment are presented below. We encourage you to read this application note that covers each of these tips in more detail using theoretical reasoning and experimental data.

• Assess HDR potential using a short insertion before attempting larger insertions. When possible, avoid using low-activity guides, as they limit HDR rates.
• Consider the position of each guide sequence relative to the desired change. The HDR rate decreases significantly when the template insertion is just a few bases away from the cut site. Therefore, the mutation site and the cut introduced by Cas9 should be close together.
• Use the Alt-R™ HDR Design Tool to design efficient gRNA and HDR donors. This tool, designed after extensive experimental testing, provides optimized HDR repair template design and Cas9 guide RNA selection.
• Carefully select DNA templates.
  • For short insertions (< 120 bp), including stop codons, protein functional sites, and detectable tags, use ssODN templates (e.g., Alt-R HDR Donor Oligos).
  • For longer insertions, use Alt-R HDR Donor Blocks that are available up to 3000 bp in length. Refer to this page for more information about Alt-R HDR Donor Blocks.
  • The Alt-R HDR Design Tool will automatically recommend the preferred donor template format based on the length of the insertion.
• Lengths for homology arms.
  • Use 30–60 nt lengths for homology arms for short ssODN donors (e.g., Alt-R HDR Donor Oligos; <200 nt total length).
  • Use 200–300 bp lengths for homology arms for longer dsDNA HDR donors (e.g., Alt-R HDR Donor Blocks; 200–3000 bp total length).
  • Use the Alt-R HDR Design Tool that can design both short and long donors and automatically selects either Alt-R HDR Donor Oligos or Alt-R HDR Donor Blocks in the output.
• Use the Alt-R HDR Enhancer V2, a small molecule compound that effectively diverts repair pathways towards HDR, successfully enhancing overall HDR efficiency.
• Consider adding silent mutations into the protospacer or PAM that play a double role of preventing the premature degradation of the dsDNA template and the recutting of the genomic target DNA. This feature is already built into the Alt-R HDR Design Tool.

To summarize the tips and results presented in this post, it is important to test different HDR experimental conditions and select the most efficient ones because of the relatively low baseline rate of HDR (especially compared to the NHEJ pathway). Select the most active gRNA design that makes a cut as close as possible to the intended insertion site. Choose carefully designed HDR templates, taking into account the insertion size, default homology arm lengths, and selection of silent mutations. Finally, use the right tools and reagents for your experiments. For more information about CRISPR, HDR, and the products offered by IDT for your experimental needs, read this application note and refer to this page.

*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations. Doc ID RUO23-1953_0011