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
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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