Why two guided RNA (gRNA) are used to develop single CRISPR mutant line?
Two cuts adjacent to each other provide more opportunity for cutting (i.e. one gRNA more efficient than other) and a larger deletion to make detection easier. Hope this helps!
NHEJ is major repair mechanism for nuclear genes, end joing repair may not form indels causing mutation and the affect of single guide RNA may not be detected. Using 2 guides makes detection easy a simple if guides are far enough to see for example with simple agarose gel
Dear Yurdagul,
Many thanks for the reply. Now I understood that why we are using two gRNA in CRISPR to knockout single gene.
Cheers,
Narendra
More guide RNA, more cleavage, high disruption of gene, efficient knockout ..tadaaaaaa(::)
How do we do most efficient HDR for knocking in in organelles with CRISPR/Cas9? How do we do?
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