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If I have some synthetic DNA sequence (<=20 bp long), is there a way for me to reliably insert this sequence next to some n-bp motif? I'd like for this to be possible in humans. If so, are there any restrictions on the size of n?

I have explored the use of CRISPR/Cas9 for this end, but it is limited in the sense that the gRNA target sequence must be adjacent to a PAM sequence. Attempts have been made to solve this problem (http://www.nature.com/nature/journal/v523/n7561/full/nature14592.html#affil-auth), but they fall short of full generality for n. Are there any better approaches?

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  • $\begingroup$ How big is the n (size of one/both flanks)? Also have a look at this post. $\endgroup$ – WYSIWYG Dec 14 '15 at 9:16
  • $\begingroup$ Just use something not restricted by PAM site, such as Zinc Finger nucleases or TALENs. But NOTHING about DNA insertion is reliable. The most likely repair route would be nonhomologous end joining, but you're looking for homology directed repair based on your 19 base template. Also 20 bases is way too short for good homology. You might be able to improve insertion rates by using a single-stranded template though. $\endgroup$ – user137 Dec 14 '15 at 14:50
  • $\begingroup$ gRNA is just for the targeting of the cut in order to engage site directed homologous recombination. You need to insert your synthetic sequence in between homology arms to get it to incorporate. $\endgroup$ – AMR Dec 14 '15 at 14:51

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