What are the advantages and disadvantages of using single-stranded vs. double-stranded HDR templates for engineering gene knockins?
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 of fluorescent cells in the overall edited population could be used as a proxy for the frequency of successful HDR. However, some subset of these fluorescent cells could have resulted from random integration of the HDR template in frame with a gene other than the intended genomic target. Such events are less probable when ssDNA is used as a template for HDR.
References
Li, H et al., Design and specificity of long ssDNA donors for CRISPR-based knock-in. bioRxiv doi: https://doi.org/10.1101/178905 (2017).
Roth, T.L et al., Reprogramming human T cell function and specificity with non-viral genome targeting. Nat. Lett. 559, 405–409 (2018).