I need to splice a gene into a human cell genome, with highest rate possible. I mean, doesn't really matter where the gene enters, nor does it matter if some cells die as a result of this.

CRISPR know to knock-in genes with very high specifically, this reduce the success rate if we have a low amount of gRNA and/or of the protein.

I need to insert the gene, without the need of targeting some specific place.

Is this possible in some way with CRISPR?

I know that there may be better technique to do this, but I can only use CRISPR.

  • $\begingroup$ Is there a good way to detect the gene product in a living cell? Because you could just sort the cells by FACS and take the cells that have the knock in, then grow those. $\endgroup$ – user137 Mar 27 '15 at 22:43
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    $\begingroup$ Could you please define what the question actually is? If it is "can you use CRIPR/Cas9 to insert a gene in a genome?" Yes you can. $\endgroup$ – cagliari2005 Mar 28 '15 at 1:55
  • $\begingroup$ @cagliari2005, The question is, is it possible to insert a gene with Crispr with high success rate, when I have a very low amounts of the Crispr in the cell? I mean I actually need to reduce his high specifically - to get higher rates. Is it possible ? $\endgroup$ – Robertos Mar 28 '15 at 8:50
  • $\begingroup$ What determines the specificity and cleavage efficiency is the guide RNA. As you cannot act on that I personally don't know any tricks on something like cell culture conditions which would do what you want. $\endgroup$ – cagliari2005 Mar 28 '15 at 17:06
  • $\begingroup$ Maybe you could look at conditions that increase homologous recombination rate. $\endgroup$ – cagliari2005 Mar 28 '15 at 17:22

A paper was published about a week ago in Nature Biotechnology and adresses your question, Maruyama T et al., 2015. I must say I found the authors' strategy extremely clever.

It is not about increasing efficiency by reducing specificity, but simply increasing efficiency (which is your ultimate goal anyway). What the authors did was to inhibit nonhomologous end joining (NHEJ) to promote homology-directed repair (HDR), two DNA repair mechanisms that compete in cells and of course HDR is the mechanism needed for the CRISPR/Cas9 system.

They achieve NHEJ inhibition using the molecule Scr7, a DNA ligase IV inhibitor which in turns perturbes NHEJ.

Using Scr7 they boosted by 3 to 19 fold (depending on the cell line) the insertion of the target gene. Here the graph showing these results

See paper figures

Moreover using 1μM of Scr7 over 24h on DC2.4 cells increased the % of transfected cells from 4.58% to 58.3%, a neat ~13-fold increase. Here their results:

See paper figures

Hopefully this should give you some ideas.

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    $\begingroup$ Interesting answer. $\endgroup$ – canadianer Apr 2 '15 at 6:09
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    $\begingroup$ Looks like they also used single stranded DNA oligos for some of their short insertions (see supplementary table 7), consistent with my speculation based answer below. The longer inserts are still double stranded. Would be interesting to see if long single stranded DNA would insert more efficiently too. Just glad to see I was speculating in the right direction. $\endgroup$ – user137 Apr 2 '15 at 14:24
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    $\begingroup$ @cagliari2005, wow, thank you!! Really great. $\endgroup$ – Robertos Apr 2 '15 at 14:39
  • $\begingroup$ Just a side-question: is it fine from a copyright perspective to publicly post diagrams from articles? $\endgroup$ – TMOTTM Sep 23 '15 at 18:59
  • $\begingroup$ @TMOTTM It is not plagiarism as I am citing the source but regarding copyright infringements I think you are right in pointing that out. As those figures comes from Nature Biotechnology I am not supposed to publicly post them (I actually removed them) - see Nature policy. If they were coming from open-source journals (say PLoS one) then you can as they are publicly available. $\endgroup$ – cagliari2005 Sep 23 '15 at 19:47

I'm going to preface this answer with the disclaimer that I have never used CRISPR/Cas, and there is a fair amount of speculation here.

But I think that efficient CRISPR mediated knock-in probably has three parts, targeting, integration, and selection.

The targeting is accomplished by the guide RNA, which is often introduced as DNA on the same plasmid that codes for the Cas9, or can be in vitro transcribed or synthesized as delivered as RNA. However, a poster suggests that a properly designed DNA fragment can be delivered and produce sufficient amounts of guide RNA in situ to produce efficient cutting.

The integration step is where the template DNA is inserted into the double strand break by homologous repair. Homologous repair efficiencies vary greatly from cell to cell, and is often less efficient than non-homologous end joining. However, using a single-stranded DNA might increase the efficiency.

However, even if only a small percentage of cells is correctly edited, it may be possible to select those cells and grow them. NEB describes a Cas9-GFP fusion (page 4) that can be used to bind GFP to a certain DNA sequence. It may be possible to produce CFP and YFP variants of this construct and use FRET to detect cells with the knock in. (This is where the speculation comes in) With the two fusion proteins and two guide RNAs for adjacent sequences in the knock-in gene, you might be able to detect cells where a CFP and YFP cas9 are close enough that you get FRET. This might allow you to sort cells by FACS, and then grow those cells.

Of course if your knock-in is making a membrane bound protein exposed to the surface, you could just use labeled antibodies to detect that protein.

  • $\begingroup$ Thank you, but I don't ask how to separate the cells. I ask how to force Crispr to insert in higher success rate. How to make him less specific or what regions to target. If it's possible. And your second link about Zinc-finger nucleases and not Crispr. $\endgroup$ – Robertos Mar 31 '15 at 8:57
  • $\begingroup$ The second link is about homologous recombination. CRISPR and zinc-finger nucleases are just about cutting the DNA, homologous recombination is how the new gene is actually inserted. And I don't think you're going to easily push the CRISPR success rate above 50% of all cells, the recombination rate will probably be lower. A good selection strategy can find those recombined cells and give you a population with 100% recombination. $\endgroup$ – user137 Mar 31 '15 at 14:04

If you want to have random integration you need to find gRNA sequences that are nonspecific ; everything built for CRISPR so far has been geared towards precise integration, not random integration.

Talk to an informatician about pulling out non-unique sequences from the human genome. You will then need to clone out a library of 20nt sequences that end with a PAM sequence (NGG) and chuck them into cells with another construct encoding a Cas9 and the insert.

Of course, if you simply need integration you don't need CRISPR either - you can use any bog standard lentiviral vector and you will get a stable cell line made with the gene inserted in random places. Using CRISPR for this overcomplicates things greatly.

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    $\begingroup$ OP says he "can only use CRISPR". $\endgroup$ – TMOTTM Sep 23 '15 at 18:57
  • $\begingroup$ Yes I am well aware; I am also aware that this is a massively complicated and probably a very ineffective way of doing things. Not least because spiking something in with a CRISPR targeting strategy often requires homology arms of up to a kb; and in this scenario, it means one has to not only make a library of sgRNAs but also a library of his insert flanked by 1kb sequences for every locus he is trying to target. $\endgroup$ – Ankur Chakravarthy Sep 27 '15 at 14:03

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