I am trying to knockout huntingtin gene from HeLa cells (human epithelial cells). I used CRISPR explorer and benchling.com to determine the best guide RNA sequence that would bind to the target DNA sequence and cas9 could then make the cut. The gRNA I have is as follows:


The next step for me is to design the primers with the following sticky ends

Forward primer: 5'-TTGTTGN(19)-3' Backward primer: 3'-ACN(19)AAA-5'

The problem I'm having is writing the oligonucleotide sequence of the primers. Since the gRNA strand is negative, I'm assuming it's a 3'-5' sequence, hence the reverse of it will be the 5'-3' sequence, which I can then just use as the forward primer. Sequentially, I can take the reverse and complement of this forward primer sequence to then determine the backward primer. Is that correct? I appreciate all your help.

Thank you!

  • $\begingroup$ Is GGAGGCCTCGGGCCGACTCG the output of the gRNA finding software? If so this sequence is most likely 5'→3'. Can you share the output file of the software. If it is an online tool then can you provide the link? $\endgroup$
    May 16 '17 at 3:49

A quick check with BLAST returned the following result:

Homo sapiens huntingtin (HTT), mRNA
Sequence ID: NM_002111.7Length: 13669Number of Matches: 1
Related Information
Gene-associated gene details
GEO Profiles-microarray expression data
PubChem BioAssay-bioactivity screening
Range 1: 267 to 286GenBankGraphicsNext MatchPrevious Match
Alignment statistics for match #1

Score           Expect  Identities  Gaps        Strand
40.1 bits(20)   0.017   20/20(100%) 0/20(0%)    Plus/Minus

The sequence you have is 5'-3' and it matches the Minus strand on the Human genome. You don't need to reverse the sequence!


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