Dear Anuj,

I have successfully used commercial CRISPR/Cas9 system from SantaCruz. I asked for the sequences of the gRNA once I bought the 3 plasmids and they kindly provided them. To answer your question, I would like to point out several aspects:

1. Efficiency of the transfection: lipofectamine 2000 worked for me. Regardless of the efficiency of the transfection, I will consider sorting the cells for GFP 24-48 h after transfection (those plasmids from Santa Cruz contain GFP cassette as well). This will highly increase your chances to get a good KO since all the cells that you have after the sorting, have been transfected with one/several plasmids. Then, I seeded them to obtain single clones.

2. The number of copies of your gene of interest in the cells that you are using: If you have more than 2 copies (which was my case), you may need to increase the number of clones to analyze in order to get a full KO.

3. Use of a selection marker: I will avoid it (although I know some people like to have Cas9-stably transfected cell lines). I do not think is necessary if you use the trick of the GFP sorting to enrich the population and you will prevent future potential unexpected cuttings made by Cas9.

4. To give you an idea of the efficiency of the process: I had 6 copies of my gene of interest in the cell line, I analyzed 48 clones and I got 6 full KO and plenty of partial KO clones.

I hope this helps,

Good luck!

Ana

Dear Anuj

The design of sgRNA sequence is vital to the success of knockout experiment, make sure that your sgRNA does include the PAM sequence at 3' end.  If you are using nickase (D10A) instead of wild type Cas9 you might need an additional sgRNA targeting the gene of your interest. However, Lipofectamine 3000 works best for cotransfection of sgRNA and Cas9 plasmids.

Musheer Aalam  

Thank you Musheer, but I am using commercial CRISPR/Cas9 system from SantaCruz. They were telling me to buy another plasmid called HDR (Homology Directed Repair) but I purchased only my gene specific CRISPR/Cas9 system. Now I have no idea of the design of their gRNA sequences. In their system gRNA and Cas9 is incorporated in single vector plasmid.

Hello Anuj,

what was your selection strategy so far? The HDR plasmid is for selection as it contains a puromycin resistance gene. If your CRISPR/Cas9 system is gene specific it probably contains the sgRNAs for your gene of interest. But you need a selection marker of course to obtain your KO-clones. A few more details on your procedure so far might be helpful.

Best,

Hendrik

Dear Anuj,

I have successfully used commercial CRISPR/Cas9 system from SantaCruz. I asked for the sequences of the gRNA once I bought the 3 plasmids and they kindly provided them. To answer your question, I would like to point out several aspects:

1. Efficiency of the transfection: lipofectamine 2000 worked for me. Regardless of the efficiency of the transfection, I will consider sorting the cells for GFP 24-48 h after transfection (those plasmids from Santa Cruz contain GFP cassette as well). This will highly increase your chances to get a good KO since all the cells that you have after the sorting, have been transfected with one/several plasmids. Then, I seeded them to obtain single clones.

2. The number of copies of your gene of interest in the cells that you are using: If you have more than 2 copies (which was my case), you may need to increase the number of clones to analyze in order to get a full KO.

3. Use of a selection marker: I will avoid it (although I know some people like to have Cas9-stably transfected cell lines). I do not think is necessary if you use the trick of the GFP sorting to enrich the population and you will prevent future potential unexpected cuttings made by Cas9.

4. To give you an idea of the efficiency of the process: I had 6 copies of my gene of interest in the cell line, I analyzed 48 clones and I got 6 full KO and plenty of partial KO clones.

I hope this helps,

Good luck!

Ana

Thank you Ana for your elaborated explanations but I am still stuck with the sorting part. I do not have sorting enable FACS and GFP antibody. Do you have any suggestion for me? I am also interested to know how you sorted out your KO population from a mixture. Please let me know.

Hendrik, I grow the cells for 24hr (approx. 50% population) then changing the media and adding 1 to 3ug of CRISPR along with 10ul of lipofectamin (which we purchased for  RNAi Max). After that leaving it 48hr undisturbed in CO2 incubator @37 degree C. Changed media if color of media. After 72hr extraction of total mRNA from the culture vessel. This is the protocol I am using. Now if find any thing wrong please let me know.

Hi Anuj,

The sorting is based on the fluorescence of the GFP that is present in the CRISPR Cas9 plasmid from Santa Cruz. There is no need of antibodies. 

The sorting enables you to select those cells that are potentially KO since they have been transfected with the CRSIPR-Cas9 plasmid. However, most of them will be partial KO (because there will be at least to copies of your GOI in the cells). So, after collecting the GFP-positive cells, I seed them to obtain single clones and screen them, using WB initially, to find a full KO.

I would strongly recommend to look for a Cytometry Facility that can provide you with a FACS machine. If that is not possible, you could seed the cells in 384-well or 96-well plates at a confluence of 0.6-0.7 cells/well 24h after transfection and manually, under a fluorescence microscope, identify the GFP-positive clones and keep growing those ones for further analysis.

I hope this helps,

Best,

Ana 

Dear MR Manwar,

When I said complete KO, I meant 'knocking out' (i.e. successfully disrupting) all the copies of your gene of interest based on the binding of the guideRNAs to their target sequences.

Hello, everyone,

I try to knockout my gene in Arabidopsis. Until now i did  not find a knock out line. I selected 16 lines. I do not know what the problem is.

Dear all,

I would like to ask about most appropriate way(s) to find full KO my gene of interest from different cell clones in case if I have not suitable antibodies for detection the endogenous protein. 

Should I start from checking mRNA and then genomic DNA or do it simultaneously?