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I need to stop some protein from being active and searching for some universal way to do so. In mammalians.

With CRISPR it is possible to knock-out the entire gene. But it's a little complicate (need two gRNA for eg.). There are also another reasons, so I need to do this with only one gRNA.

So I am thinking about inserting some mutation.

My question: Is there way to make some mutation that will prevent creation of functional protein, with high efficiently - with one gRNA. And without need to know the active site of protein.

For example, frame-shift or mutation around start codon? Is it possible? If so, how can I do this? To where I need to send the gRNA?

Thanks.

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    $\begingroup$ First of all, If you talk about system, please specify if it is cell line or animal. Secondly, if you say "There are also other reasons", specify them too. Also, what do you mean by "without need to know the active site of protein"? $\endgroup$ – Oct18 is day of silence on SE May 7 '15 at 7:32
  • $\begingroup$ Well, synthetic CRISPR/Cas systems do have an option of having a single guide RNA (sgRNA) $\endgroup$ – Kunal24 May 7 '15 at 8:38
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You can do with a single gRNA. All that CRISPR-Cas, ZFN or TALEN systems do is to introduce a double strand break at a specific site. The DNA gets repaired via two mechanisms — non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is error prone and it may introduce indels that can compromise with gene function (frameshifts etc). While HR can be used to integrate a transgene or repair a mutation, NHEJ can be used to create mutations.

I haven't really knocked out a gene using CRISPR-Cas but it is good to target the initial part of the ORF. There will be many other factors too that will determine what site should be chosen for targeting and an important factor would be the uniqueness of the target sequence. See this site for guidelines on selecting a good target sequence.

You would have to do rigorous screening nonetheless; the advantage of insertional knock out is that screening can much be easier (for e.g. by inserting GFP or puromycin-resistance gene).

You can have a look at this paper and the supplemental information; they have targeted 18080 genes with a CRISPR-Cas library.

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  • $\begingroup$ Huge disadvantage of insertion is very poor successful rate. Less then 5%. So I still looking for the answer. Where in the gene to cut, to make protein not working. About ORF, I wrote about start codon, but need more information and sources. Where exactly. Thanks. $\endgroup$ – Robertos May 1 '15 at 14:04
  • $\begingroup$ And about the uniqueness, off topic question, is it possible to make a gRNA that will target a lot of different places in genome? Didn't heard about that. Nonspecific gRNA? $\endgroup$ – Robertos May 1 '15 at 14:07
  • $\begingroup$ @VGranin the insertion rate you refer to is inserting a specific sequence, a non-specific indel mutation to cause a frameshift like WYSIWYG is talking about can have success rates upwards of 70%. And the only way you could target a lot of different sites with a single gRNA is if all the sites had a sequence in common, gRNAs are specific to a given sequence. $\endgroup$ – Luigi May 1 '15 at 14:40
  • $\begingroup$ @VGranin As I said you can use a single gRNA to make the cut. Definitely ahead of the start codon but not too far off (if frameshift on non-sense mutation happens then you will just have a little bit of the original sequence). So you have to optimize between closeness from start codon and other factors such as unique site and others mentioned in the link that I have given in the answer. $\endgroup$ – WYSIWYG May 1 '15 at 14:48
  • $\begingroup$ @Luigi. Thank you. But he mention a GFP insertion... Anyway, what CAS9 do is just making break. A non-specific insertion it is not something that we do, it's the cell way to repair. What I need, is a place to target gRNA to make a break that will make protein non working. $\endgroup$ – Robertos May 1 '15 at 14:49

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