Dear all,
Me and a colleague have come across the question whether a single guide RNA is enough to sufficiently delete e region (let's say a complete KO), for which the two homologous regions are ~1-2 kb apart. If the guide is close to the end of one HR arm, it will be distant from the other, so will this be efficient enough?
Zhang lab advises that the two HRs should not be more than 100 bp apart, however some papers show that it is possible and efficient if the distance is larger. Based on your experience, I would like to ask you two questions:
1) is one gRNA enough when the 2 HRs are > 100 bp apart (e.g. ~1-2 kb)?
2) if not, and two gRNAs are needed, would you advise transfect (electroporation) 2 separate plasmids, one for each guides or use a plasmid with tandem gRNAs (e.g. pX333)?
Thank you for your help! :-)
Best,
Ana Pena
Ana
Looks that you want to insert something in the middle of a gene to knock-out it. If yes.. a single gRNA should be enough, but you need to design the gRNA close to the middle region of the recombination area.
Of course 2 gRNAs will give you better results, as far as you can eliminate a big fragment of DNA.
For transformation of the gRNAs tandem systems are OK, but if the transformation efficiency of your cells is high you can also think about using the synthetic gRNA directly, without the need of expressing it.
Hope it helps. Best
Hi Francisco, thank you for your reply! In my case I want to substitute an entire coding sequence by another, and the strategy we were doing was using only one guide and 2 HR that were quite apart (HRs are respectively the 3' and 5' end of the original gene, which are more than 1kb apart). The guide is in the middle of the original gene, but much closer to HR1 than HR2... I guess the efficiency will be lower than if using 2 guide RNAs... but we will give it a try... I did a drawing and attached it :-) if you can give it a look it will be great.
Thank you,
Ana
Hi Ana,
Any update in your CRISPR experiment?
I am planning the exactly same experiment...
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