I am interested in interrogating four candidate transcription factors for their role in a mouse disease model. To do this, I would like to independently knock out these factors in bone marrow-derived cells in vivo. Instead of breeding several knockout lines, I thought to generate bone marrow chimeras in which the donor bone marrow has been edited using CRISPR/Cas9 to knockout the gene of interest. Does anyone have experience with CRISPR/Cas9 in hematopoeitic stem cells? Any suggestions on lentiviral tranduction vs electroporation vs nucleofection of the Cas9 and gRNA plasmids? Thank you in advance for you help.
For bone marrow stem cells specifically, you'll need a more powerful reagent. We've used this kit (https://altogen.com/product/bms2-transfection-reagent-bone-marrow-cells/) for bone marrow stromal cells, but if you're using primary cells you'll probably need this one (https://altogen.com/product/altofect-transfection-reagent/). The kit we had came with a CRISPR/Cas9 protocol/information sheet. I've attached it to this answer as well.
Hi David, A similar question was asked before which I answered. We too are doing CRISPR/Cas in BM progenitors. We have tried nucleofection and electroporation of lin- cells and we have had real problems getting enough vector in - especially as the cas9 vector is so large. We made retroviral vectors - two different one where one was cas9 IRES mCHERRY and a guide RNA vector with GFP. But again, the titre of the cas9 was very very low. We also tried the Neon system, and this was better, but still very very low efficiency.
So now we have ordered and just received the cas9 mouse and are using BM progenitors from this with a small guide RNA vector. We have only begun but hope this will overcome the bottle necks we have so far seen. What we have found that stable expression of guide RNAs leads to both alleles often being targeted. The drawback being in this case that is complete KO is lethal. Electroporation in this case would be better as then you could get haploinsufficiency which may better model what you are trying to see.
Anyway, good luck with your expt and if you do manage to find a good protocol for high levels of electroporation/nucleofection into lin- cells, let me know.
https://www.researchgate.net/post/Are_human_CD34_cord_blood_cells_amenable_to_gene_disruption_by_CRISPR_Cas9
Hi,
I would have the same question- did the cas9 mouse work? Thank you,
Dear Charles E. De Bock,
It would be very interesting to know if the Cas9 mouse have solved your problem of getting Cas9 expression, and if you can now do genome editing in BM-progenitors.
Thank you very much in advance for your help!
Best,
Karine
I'll offer the general suggestion that people consider using RNP:
https://www.jove.com/video/57350/enhanced-genome-editing-with-cas9-ribonucleoprotein-diverse-cells
Article Selection-free genome editing of the sickle mutation in huma...
Same thing as Ross, RNP delivery may help. And I would suggest the new Viromer reagent we have developed for a safe and efficient transfection:
https://viromer-transfection.com/crispr
And by coincidence, I read this blog some days ago, maybe interesting for
you to contact this team in NL
https://www.synthego.com/blog/crispr-conversation-with-rasmus-bak
BR,
Sandra
For bone marrow stem cells specifically, you'll need a more powerful reagent. We've used this kit (https://altogen.com/product/bms2-transfection-reagent-bone-marrow-cells/) for bone marrow stromal cells, but if you're using primary cells you'll probably need this one (https://altogen.com/product/altofect-transfection-reagent/). The kit we had came with a CRISPR/Cas9 protocol/information sheet. I've attached it to this answer as well.
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