I have one more doubt about crisper cas9 experiment,
I generated few transgenic plants for crisper cas9 mediated mutation, When I analyzed sequence for editing/mutation by sequencing I found that crisper cas9 has digested DNA at downstream of PAM site (5-6 bp after PAM site, single base pair deletion). I just wonder, Is it correct mutation? Is it possible
Generally Crisper cas9 edit the DNA at upstream of PAM sites (3bp upstream of PAM site.
Can anyone please guide me about this??
Hi Harshraj,
That is wield. It is supposed to be upstream. But, how many edited plants in your hand showing they got edited downstream of PAM? Or, it is just a single plant and a single event?
Attached a picture to show the cut position.
Is any similar sequence around the 'targeted' sequence, which made the sgRNA mistakenly bind to it and caused an off-target cut?
Thank you Dr Yuan for responding. its just single plant showing editing after PAM site. But there are few research papers published in Nature communication showing editing of sequence after PAM site. please check the attached file.
Hi Harshraj,
Where in the paper it says that ("editing of sequence after PAM site")? They just presented that way so the sequences looked like edited after the PAM.
See attached picture, if I turn around the picture, you will see the upper DNA strand is 5'......CCN.NNNNNNNN.........3', which matched Gao's sequence presentation order that was presented as: ....CCT.NNNNNNNN......
Does this make sense to you?
Also, do you have other edited plants? and are they been edited upstream of PAM?
By the way, how come you have only one 'edited' plant (although I am not sure that one is really edited)?
Use this technology, usually people will obtain a 'population' of edited lines, which have different numbers of inserted bases or deleted bases at repaired DSB site, just like those described in the paper you attached.
Thank you Yuan for detail information. I think its false editing, I am analyzing another bunch of transgenic plants now. lets see
Yes, please update us when you have new results, Harshraj. Thank you for sharing your research with us.
Hi Harshraj,
I have a couple of thoughts:
1. No matter how this 'mutation' comes from, can this 'deletion' cause mutation of the gene of your interest? If so, you still have a knock-out line ready for analysis. However, you might have a hard time to explain to paper reviewers how this mutation formed.
2. Let's don't treat this mutation as 'false' editing (as you claimed) yet, we just temporarily put it on the back burner and wait. it might be a very rare true event, we just don't know now. This can be a new discovery, who knows. New discovery can happen every day in science.......
Best,
There is a possibility. Not neccessarily due to editing per se but can be due to re-arrangements.
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