I got the sequencing result for CRISPR knock-out experiment, but guide RNA did not show any mutations but the PAM sequence showed a single substitution. However, the sequences flanking the guide RNA and PAM sequence, showed deletions, insertions, and substitutions. I want to know whether this result can be said as edited?
Hi...
It is possible that the CRISPR-Cas9 system was able to edit the DNA at the target site, even though the guide RNA sequence itself did not show any mutations. CRISPR-Cas9 works by using a guide RNA molecule to direct the Cas9 enzyme to a specific location in the genome. Once the enzyme is at the target site, it can make cuts in the DNA, resulting in deletions, insertions, or substitutions.
If the sequences flanking the guide RNA and PAM sequence show deletions, insertions, and substitutions, it is likely that the CRISPR-Cas9 system was able to make edits at the target site. However, it is important to carefully analyze the sequence data to confirm the presence and nature of the edits.
In some cases, the CRISPR-Cas9 system may not make any edits at the target site, even though the guide RNA is correctly targeted. This can occur if the DNA at the target site is resistant to cutting or if the repair mechanisms of the cell are able to accurately repair the cuts made by the enzyme.
I hope this information is helpful.
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