Hello,
I'm designing a CRISPR/Cas9 protocol for school purposes (I attend a extra project next to my study). I have to use the pCas9 plasmid (addgene #42876) for CRISPR eventually. This plasmid contains a separate tracRNA site and a separate crRNA design site. I did a literature study on how to construct a crRNA and got the standard 19-20 nucleotide next to PAM-site. However, I noticed in the protocol on the addgene site of de the pCas9 plasmid that you have to make a oligo that's 30 nucleotides long for constructing the spacer sequence. It has to be in this format:
5’- AAACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNG -3’
I understand that the repeats that around upstream or downstream of the crRNA location anneal to the tracRNA of the plasmid. I'm a little lost know and don't get my anwers after some literature study. I hope someone can help me how I can construct a crRNA for the pCas9 plasmid.
Regards,
Jesse
https://info.addgene.org/download-addgenes-ebook-crispr-101-2nd-edition
should explain it all.
gRNA = crRNA+tracrRNA linked by a GAAA loop
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