Hii everyone,
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?
It seems that your design might knock out your GOI and replace it with a GFP-DsRed. If you want to fuse the GFP to the protein coded by the GOI, you need to insert the GFP just upstream of the start codon of the GOI. So, your right homology arm (RHA) should contain the CDS of GOI. The attached pictures may be helpful to understand the principles. Please note that it is not necessary to include the intron to RHA. It just need to reach the desire length (~1 Kb is OK). The sgRNA ideally should target just close to the ATG of GOI (distances more than 25-30 bp will decrease the efficiency).
@ Mohsen: I dont think the donor template ( HA---GFP---HA) will still knock out the GOI, its just that there will be 200bp between GFP and GOI. Genome sequence might look like this--> Upstream 800bp HA----GFP---200bp non coding region----Endogenous GOI
Alternatively, is it possible to design a donor vector as: HA---GFP:GOI---HA with a gRNA targeting region close to ATG of GOI.
As discussed by other members, I thing its best to leave endogenous 5' upstream regulatory regions intact with least intrusion ( includes kozak sequence ? ) making a nick close to ATG region.
Hii,
Thank you for the info.
I am working with fruitflies.I understand that it is necessary to design a gRNA which targets region close to ATG of GOI, but what if we cannot design a gRNA close to that region?I am using flycrispr website to design the gRNA but couldnt get one that targets desired region.
@ Vijay: I'm concerned about Razia's design because the schema of genomic locus provided by her (Homology arm(1.2kb)--GOI--Homology arm(1kb)) shows that she has picked the right homology arm from the downstream of GOI (maybe 3'UTR) which will undoubtedly lead to knock out and replacement of GOI CDS by the GFP-DsRed cassette. Actually, it is a typical design for gene knock out!
I assumed that she wanted to fuse GFP to the N terminus of GOI protein. In this case, that 200 bp non coding region can be a problem.
However, I agree that your alternative design can work. The only concern is the efficiency, which may be affected by the distance between the DSB and the right homology arm (including GOI).
@Razia: I don't recommend, but if you want to insert the GFP 200 bp upstream of the ATG, you should make sure that: (1) GFP will be in the same frame as the GOI, (2) the stop codon of the GFP is removed, (3) and there is no stop codon in the 200 bp spacer between GFP and GOI (which is unlikely!). In spite of these considerations, this spacer may still cause some problems due to coding a ~ 65 aa linker peptide with unknown characteristics.
The other possibility is to use your 200bp upstream sgRNAs along with another sgRNA downstream of the ATG. Then you will have two DSB each in one of the homology regions. With this strategy, you can use a targeting vector similar to that in the picture in my previous post.
@Mohsen: Thank you for the insights.But my concern is what if we cant get gRNA that targets the above mentioned regions?
Is Drosophila genome best annotated only through flycrispr website ?
If that isnt the case you might want to try the top common gRNA designs websites ( crispr.mit.edu and Harvard's chop chop selecting fly genome - dm3 ).
You can feed ~300 bp that include just 100 base pairs upstream of ATG site of GOI. Look for top gRNA hits with least off-targets, choose a gRNA if it is closer to ATG irrespective of its rank being lower ( eg.Rank 4, 5 ) than another gRNA (eg.Rank 1,2) which cuts farther away from ATG. Just mind the number of off-target regions though.
gRNA designs do depend on the target genome including availability of PAM sequence. One thing I am unsure which you can discuss with your lab members :
- Will the gap (200bp) between GFP and GOI contain any critical regulatory regions for the GOI , as this is a fly genome that is technically shorter and more gene-dense than a human genome.
If gRNA choices are limited, you can still try TALEN ( cumbersome though ! )
Good luck !
@Mohsen: Oh yes, you are right ! Sorry, my mistake. Silly me, I understood that the left homology arm is 1kb upstream of GOI, but was wrong in assuming that the right homology arm was flanking the Cas9 cut site, instead it is downstream of GOI. Your design sounds right .
If you have idea, N terminal fusion of GFP to Cas9 will create any problem in the functionality of Cas9 protein?
Hi Razia,
We recently generated GFP or mCherry reporter cell lines by knocking in the flurophore at the N-terminal. We used one gRNA; Our gRNA edited the 20 nucleotide sequence around ATG, so that was convenient.
In a very non technical way, I would say you need to visualize how the gene is supposed to look post KI; Use this to design your donor template (see the attached pic). It is not ideal to have a gRNA cut 200bp upstream of where you wish to insert you transgene (max recommendation is ~50 bp)...As others have pointed out, the efficiency of HDR will be much lower.
Could you use Cpf1 or any other enzyme to cut closer to ATG?
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