Splice in with CRISPR/Cas

Question

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.

Answer 1

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.

Answer 2

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.

Answer 3

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.