To synthesize SgRNAs for RNP CRISPR - we have to design 20 bp targets based sequences, but what about the sgRNA scaffold ..is it species dependent?; Secondly, is it cas9, dcas9 or nickase dependent? ; Thirdly, for cas9 n (nickase), paired sgRNAs how to get it done if we have with us only the designed sequences targeting the sites...
Kindly throw some light on scaffolds ...for ordering the synthesis of SgRNAs.
Hi Pragya Gupta ,
Your questions regarding sgRNA scaffolds:
Q1. Is it species dependent?
Ans. Yes. It depends on the microorganism Cas9 was derived from.
Q2. Is it dependent on various derivatives or Cas9?
Ans. No. The scaffold will remain the same for all iterations of the same species derived Cas9.
Q3. How to get the Cas9 nickase done?
Ans. You must have two sgRNA sequences flanking the target region. For better insight, I would suggest you to read this: https://blog.addgene.org/crispr-101-cas9-nickase-design-and-homology-directed-repair
Hope it helps!
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