I'm a student started who has started learning about CRISPR/Cas9. As I understand it, CRISPR/Cas9 is an enzyme that is used to cut a gene at a specific sequence. I would like to know how scientist do the next step to insert/edit a genome.

For example, say I have an original sequence ...AAATTT... Is it possible for me to insert a new sequence into the middle so it becomes like this: ...AAAGCGCGCTTT... ? Or is it possible for me to edit a single nucleotide of the sequence like this : ...AACTTT... ?

How is a new sequence inserted at the cut region? Is there any enzyme required to make them join each other? How is it ensured that the sequence is not inserted or joined at the wrong position ?

  • $\begingroup$ youtu.be/2pp17E4E-O8?t=1m50s $\endgroup$ – Dale Aug 9 '16 at 17:23
  • $\begingroup$ @Dale thank you for this video, i looked at the video but don't clearly understand, does it mean that the cut make a sticky end instead of blunt end and we need to design a sticky oligo so they can join each other? And as i see on the video, no enzyme is required for joining them together. Can you explain more a bit? $\endgroup$ – joe Aug 10 '16 at 2:26
  • $\begingroup$ @Dale As i known, to join 2 sticky end dna together, it seems that we need to use an enzyme called 'dna ligase', isn't it ? $\endgroup$ – joe Aug 10 '16 at 3:15

Cas9 is used to create a double-stranded break (DSB) in genomic DNA. The cell can then use homology directed repair (HDR) to repair the break. The cell can also use non-homologous end joining (NHEJ) to repair DSBs, but this isn't useful for inserting specific sequences. In HDR, a sequence homologous to either side of the break site is used as a template so that the break can repaired error free. Naturally, the homologous template could be a homologous chromosome or replicated copy of the broken chromosome.

However, consider a situation where you introduce DNA into the cell with regions of homology to the break site and whatever other sequence(s) you wish to insert/delete/change. In this case, the break can be repaired using this exogenous DNA as a template and your changes will then be incorporated into the genome.

There are other methods of introducing site-specific DSBs to the genome, such as TALENs. The big advantage of CRISPR/Cas9 is that site recognition is performed by RNA rather than protein (it is much easier to synthesize small RNAs than to engineer large proteins).

  • $\begingroup$ While this answers covers all of the OP's points, it might be worth to point out a few more specifics. (i.e. which proteins are needed for HDR or the probability of successful target integration) $\endgroup$ – Nicolai Jun 7 '17 at 5:51
  • $\begingroup$ @Nicolai More detail is always good but, given the lack of interest in this question and answer, I'm not sure it's warranted. I encourage you to post your own answer or edit this one if you have more information to add. $\endgroup$ – canadianer Jun 7 '17 at 6:15

There are multiple Cas9 proteins so much of the information is specific to a particular protein for the specifics.

Inserting genes in the point cleaved by CRISPR is definitely possible--of course this requires other proteins. Cells often contain those proteins, so complementary sequences on the ends of the insertion sequence is technically all that should be needed for insertion inside cells. There are many details to all this, but that is not what your question is asking about.

A single point mutation has recently been edited: however it was not inside a cell: https://www.statnews.com/2016/04/20/clever-crispr-advance-unveiled/


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