I would like to use two or three sgRNA to KO target gene using CRISPR technology, these sgRNAs need to target the different exons, how distant between the sgRNAs would be good to efficiently KO the target gene? like sgRNA1 targeting Exon2, and then sgRNA2 targeting Exon4 or 5, is this good or not? Anyone knows this question, please give me answer. Thank you so much!
While I can't really speak toward effects of spacing out the targets, the most straightforward way to ensure high edit efficiency is to find high-efficiency sgRNAs across all exons. This can be done using a predictive tool such as http://crispr.wustl.edu/
Some additional things to consider are to make sure the target sequence is not at the end of the gene, and that you may have to use up to 3-6 different targets to have a good chance of successful editing.
Hope this helps.
Thank you very much for your informative answer! One more question: why we can not put the target sequence at the end of the gene. thanks!
You're very welcome!
For your follow up question - it has to do with the effects of Cas9 cutting rather than the cutting efficiency itself. Even if cutting is successful there is a lower chance of gene disruption if the cut happened close to the end of the gene. To my understanding the concept is that the majority of the protein would still be transcribed correctly in this case.
Reference:
"As expected, we observed diminished activity of sgRNAs targeting close to the C’-terminus, since frameshift mutations close to the end of a protein are less likely to disrupt expression "
Doench JG, et al. Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation. Nat Biotechnol. 2014;32:1262–1267.
Hi./ Wang
Greetings!
I think the distance between two sgRNAs should not be less than 50 nt mostly 100 nt as distance between two sgRNAs works well, higher efficiency can be obtained if the two sgRNAs target the two strands one sgRNA target sense and the other target the antisense strand
Best of Luck
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