Hi to all! I'm entering the field and so I have a lot of question..
The first one is the model: We are currently working with a cell line that is Hypertetraployd , and I'm wondering if it could be possible to do HDR on this cell lines. As I understand, the system does not have an allelic preference. So, in your opinion could be possible to use this system or I have to switch to another model? Another conceptual question: How the Cas9 recognize preferentially a ssOD instead of the other allele? or it is not? so the frequency of mutation would be quite low?
many thanks for any input....
What is HDR? Homologous recombination?
It depends what you try to achieve, if you just want a modified gene expressed and not a 100% replacement of all copies I see no problem. But if you try for a 100% change I would not use your cell line, the probability of modifying two alleles is already decreased (target probability, probability of repair failure and cell death activation), having a higher ploidy is making it worse.
The guide RNA bound in Cas9 (secondary structure and sequence) recognizes the target DNA of the chromosome based on Watson-Crick pairing. The off target effect number with nickase modified Cas9 is significantly lower than original Cas9 (which cuts as one molecule) so I would recommend that latter.
http://www.nature.com/nmeth/journal/v11/n4/full/nmeth.2857.html
Thanks Ulrike, that is a very clear answer..
HDR yes is homologous recombination..I'm trying to insert a mutation into a gene to obtain a disease model, so I'm aiming to a 100% replacement ...I think I would switch to another cell line
anyway my general question remains...in theory then to be sure of modifying both alleles I should make 2 rounds of cas9 modification?
and thanks for the paper..it is interesting..the authors however worked on a X-linked gene, more easy to modify...
they did it in wheat see doi:10.1038/nbt.2969 nature biotechnology
simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew
Sequence-specific nucleases have been applied to engineer targeted modifications in polyploid genomes1, but simultaneous modification of multiple homoeoalleles has not been reported. Here we use transcription activator–like effector nuclease (TALEN)2,3 and clustered, regularly interspaced,
short palindromic repeats (CRISPR)-Cas9 (refs. 4,5) technologies in hexaploid bread wheat to introduce targeted mutations in the three homoeoalleles that encode MILDEW-RESISTANCE LOCUS (MLO) proteins6. Genetic redundancy has prevented evaluation of whether mutation of all three MLO alleles in bread wheat might confer resistance to powdery mildew, a trait not found in natural populations7. We show that TALEN-induced mutation of all three TaMLO homoeologs in the same plant confers heritable broad-spectrum resistance to powdery mildew. We further use CRISPR-Cas9 technology to generate transgenic wheat plants that carry mutations in the TaMLO-A1 allele. We also demonstrate the feasibility of engineering targeted DNA insertion in bread wheat through nonhomologous end joining of the double-strand breaks
caused by TALENs. Our findings provide a methodological framework to improve polyploid crops.
Thanks Didier, very interesting answer/paper..I've read it a first time and it seems feasible...however I'm not an expert on the DNA repairing mechanism in plant..and I guess they would be quite different than in mammalian cells. Uhm..I'm thinking to switch cell model anyway, because it seems to be a little too difficult...
F zhang on the google group (https://groups.google.com/forum/#!forum/crispr) i advice ou to have a ook at this group.
claim that usually, the two alleles are targeted simultaneously...so it is likely, three or four will also be targeted
Thanks Didier, I have also posted the same question in the g-group, but still no answer..it is anyway quite an interesting group..thanks for the suggestion!
For your gene of interest do you expect this to be a dominant or recessive mutation? If dominant you don't actually need to hit all alleles. For HDR, its common to see 4-5x more heterozygous (monoallelic) inserts than homozygous (biallelic) so it may be challenging to hit all 4 sites. You could try two rounds of gene-editing though. It'll take you a bit longer but should be doable. Also, as mentioned above if you can use the paired nickase approach to minimize off-targeting that would be ideal.
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