I am working with human ES cells and interested in inserting a GFP and RFP reporter downstream to both the alleles of my gene of interest to monitor its expression. I am thinking to keep 1 kb homologous regions and design guide RNA which cuts just before the stop codon of my gene of interest. I am seeking further guidance from any experienced person who can share the experience, provide some key references and tools.
Hello,
the method you are talking about is the classical method (1kb homology on each side, with the insert in the middle).
The site for the guide RNA double strand break is a few base pair flexible (you don't have to cut right before the "stop", but you can make it a few base pair beside if needed.)
You will find a lot a HR plasmids on addgene; so you can see the precise construction and design.
Maybe you can design 2-4 sgRNAs for the same gene: it seems the HR efficiency is highly dependent on the efficiency of the double strand break induction. So design a few sgRNA for one site, and test them into your cell to know which one induces the more the double strand break.
Did you consider incorporating a resistance cassette in order to select cells with integration of the casette. If you design piggybac or Cre sites you can excise the cassette later.
Hi Filip,
Yes, I have considered it and will use LoxP sites. Thanks!
Hello Shafqat Ali Khan,
May I know the size of your construct that you tried to insert by HDR. How many gRNAs did you use and were they targeted at the same site or different sites?
Please let me know the outcome of your work.
Thanks..
Hello Razia,
I am using a knock-out/in approach to insert to insert 5.5 kb cassette (promoter+reporter) in HPRT1 gene. HPRT gene is used for selection against 6-GT selection. I am keeping the length of homology arms as 1.5 kb. At present, I am done with strategy and beginning with cloning. I will come to know the results after about 2 months only.
Wait till then :)
Hello Shafqat,
Did you design the gRNA which targets downstream of the gene before the stop codon?If so, how many bp before the stop codon.
Thank you
@ Razia. My aim was to disrupt the gene so gRNA are designed for exon 2. In my case reporters will be expressed with their own promoter. I understand that you are interested in expressing the reporter with the endogenous promoter and that can also be done but this is a bit tricky. We could discuss it if you explain you experiment.
Hii Shafqat,
I want to tag GFP to a gene of interest (GOI) at N-terminal domain rather than at C-terminal using CRISPR-Cas 9 system. I have designed a gRNA which targets a region 200 bp upstream of GOI and homologous flanking regions 1 kb down stream and 1kb upstream of GOI. The construct looks like this
Genomic sequence = Homology arm(1.2kb)--GOI--Homology arm(1kb)
Construct = homology arm(1.2kb)-GFP-dsred-Homology arm
Since the gRNA we designed target the sequence 200bp upstream, I was wondering whether HDR will incorporate the insert correctly and does it give a fusion protein, as there will be 200bp non-coding region in between GFP and GOI?
Thank you
I think, I could suggest you a better strategy but for that, I have to do some drawing. So, wait for few hours.
Maybe this is a better solution. In this approach, you pick the best gRNA in exon 1 of you GOI. Your homology arm will correspond to the flanking region of exon1 of you GOI. In this way, you replace the exon 1 with you insert (reporter gene + exon 1 of GOI). I think this will avoid any chances of frame shift and will give you a tagged protein driven by the endogenous promoter.
Discuss among your group and let me know your views as I am also learning.
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