What are optimal lengths for HDR template homology arms?

It has been demonstrated that there is an exponential relationship between homology arm length and knockin efficiency, with ssDNA templates including homology arms 350–700 nt in length providing optimal performance (Li et al. 2017). In our own studies involving hiPSCs, optimal performance was observed with 350-nt homology arms, and the use of longer arms did not substantially increase knockin [...]

With ssODN or ssDNA, is it advantageous to use HDR templates that are homologous to the sense or antisense strand at the genomic target region?

Currently, there is no clear rule regarding the preferential use of sense or antisense ssDNA for HDR templates longer than 200 nt. However, in the case of shorter single-stranded oligodeoxynucleotide (ssODN) templates, template polarity has been found to have an effect on efficiency (Bollen et al. 2018). References Bollen, Y., Post, J., Koo, B. & Snippert, H.J.G How to create [...]

How do ssODNs differ from ssDNA? When is it better to use one vs. the other?

Single-stranded oligo deoxynucleotides (ssODNs) are produced routinely via chemical synthesis and can be used for engineering smaller edits (e.g., single nucleotide substitutions or shorter insertions <50 nt) as HDR templates with a total maximum length of 200 nt. For engineering larger insertions that necessitate HDR templates >500 nt, we recommend the use of ssDNA that can be produced [...]

What are the advantages and disadvantages of using single-stranded vs. double-stranded HDR templates for engineering gene knockins?

It has been demonstrated that the use of ssDNA results in lower toxicity and reduced frequencies of random integration relative to dsDNA (Roth et al. 2018; Li et al. 2017), benefits which can be especially important when working with difficult-to-engineer cell lines. For example, if you are seeking to fuse a fluorescent protein to an endogenous gene expressed in your target cells, the percentage [...]

How do I design my HDR template?

The sequence of the HDR template should match the intended sequence of the targeted genomic locus, including the desired edit flanked by 5' and 3' ends that share homology with the wild-type genomic locus. (These 5' and 3' sequences are referred to as ""homology arms."") Sequences corresponding to desired edits (insertions, substitutions, etc.) should be positioned in the middle of the HDR [...]