Hi, I'm currently learning the art at Vanderbilt University, specifically working with zebrafish. I think what you should do first is try different concentrations of Cas9 also use the sgRNA that phenotypically whose effects are visible, such as TYR.

I hope this helps.

Hi Raul, thank you for your reply. I am actually considering of expressing cas9 in an inducible manner in my target organism and then transfecting them with the sgRna, which targets a fluorescence protein. The disappearance of the signal will be the readout to indicate if the knockout is successful. However, I saw in the literatures that the success of knockout via this approach is currently about 30% and hence I am wondering if it would be even harder to do a double knockout on one try.

From the literature it seems like you'd have to screen on the order of 100 clones to find a homozygous KO.  But that's best case.  There can be a certain... positivity bias in the literature.  

If your cell type is clonal, you can always fall back to doing it in 2 steps.  (meaning pick a hetero after the first round, grow it up, and then do it again to make homos)

Hey John, I agree with you that a second round of transection would increase the chances of the obtaining a homo. I am just wondering if the crispr approach can make it more efficient such that a single transection will do the job. Since it requires an order of 100 clones, I guess that might be hard. Thank you for your discussion.

Hi, according to my experience, if you design good gRNA for your CRISPR, the efficiency could be really high. In my case, I did site-specific knock in, in which I co-transfect CRISPR with a donor vector. After drug selection and limited dilution, one gRNA gave me 100% recombinaiton. The other one gave 1/23. The efficiency I am talking about here is about three alleles simultaneously.  My B cell lymphoma cells have three alleles of my target gene. You can use donor vector for KO too, for example replace your gene with a GFP marker which will help you to select targeted clones.

Hi Jianliang, that's really interesting. However, may I know if cas9 was transfected as a protein or plasmid in your experiment? It is really exciting that you can have a simultaneous triple knockout and the reason why I consider to inducibly express cas9 is to ensure successful double knockout in a large population of cells. Since gRna design is limited by a specific length, do you have any other approaches that increases the specificity of its effect? Thanks.

Hi, I co-transfected CRISPR and doner vector both in the form of plasmid. I know someone transcribe CRISPR in vitro to mRNA and then transfect the mRNA into cells. The idea is that mRNA will degrade quickly so you will have less chance for off target. Thus you will have higher specificity. And also in my case, I am using Cas9n which is a nickase that only introduce a nick on one strand of DNA. The nick will be either repaired faithfully or, when you have a donor vector, enough to initiate homologous recombination. So even if your gRNA do not have very high specificity, the Cas9n will only make a single DNA break at the off target, which probably will be repaired correctly. If you do not want to use donor vector, just use two gRNA-Cas9n to introduce two nicks at the two opposite strand of DNA close enough at your gene, then the piece of DNA in between the nicks will be excised out. NHEJ will connect the break ends of your gene and you get your knockout cell.

Hi, that is a very interesting way of countering off target effects. Just two last questions before I begin testing them out. Were the cas9 and donor vector transfections you performed stable or transient? The reason why I asked this is because I wonder how efficient the transfection is. Also, will long stretches of homologous sequences be required for cas9 induced recombination or will an adaptor with short homologous sequences be suffice for the recombination to occur? Thank you once again. 

Hi, it was transient. I used electroporation to introduce plasmids into my B cell lymphoma cells and the efficiency was low because they are suspension cells. But there is GFP and puromycin selection marker in the donor vector so I can select the targeted cells with drug and GFP fluorescence. Then after limited dilution I got homozygous clones with very high efficiency, which you can not get without selection. So I suggest you also use donor vector for making konckout cells. You don't need to use long homologous arms in the donor vector in CRISPR system. 800-1000bp is enough. Someone even use 500bp. This is a really important advantage of CRISPR.

But maybe I am lucky to have homozygous clone by only one round transfection. Sometimes you may need a second round transfection and selection. 

Hi, Jianliang:

I am using HEK293T cell line and found it is very difficult to obtain homozygous clone since this line is polypoidy. Do you know and dipoidy or hapoidy lines available? For KI GFP or mCherry into a target gene, what donor vector are you using. How long are the flanking sequences required? I am trying to PCR an mCherry cassette with a Neo marker using a long pair of primers (80 bp for left and right flanking sequences)  

Hi everyone,

very nice conversation.

Does anyone know whether it is possible to select TRANSIENTLY co-transected cells with both Cas9/Nemycin and Donor DNA. Zeocin vectors?

Thanks a lot

Hi everyone,

very nice conversation.

Does anyone know whether it is possible to select TRANSIENTLY co-transected cells with both Cas9/Nemycin and Donor DNA. Zeocin vectors?

Thanks a lot

Dear esteemed colleague,

I hope this correspondence finds you well. Your inquiry regarding the efficiency of CRISPR-Cas9-mediated knockout in diploid organisms is an area of significant interest and ongoing research within the scientific community. The CRISPR-Cas9 system has revolutionized genomic editing, offering a versatile tool for manipulating genomes with precision. However, the efficiency of inducing knockout mutations can vary widely, influenced by several critical factors.

In diploid organisms, achieving biallelic knockout (i.e., disruption of both gene copies) is essential for completely abolishing gene function. The efficiency of achieving biallelic knockout is generally lower than monoallelic knockout due to the need for simultaneous editing of both alleles. Efficiency rates can vary widely, with reports ranging from a few percent to over 90%, depending on the factors mentioned above.

It is also worth noting that recent advancements in CRISPR technology, including the development of high-fidelity Cas9 variants and improved sgRNA design strategies, are aimed at increasing the efficiency and specificity of CRISPR-Cas9 editing in diploid organisms.

In summary, the efficiency of CRISPR-Cas9-mediated knockout in diploid organisms is influenced by a complex interplay of factors, including sgRNA design, Cas9 activity, delivery methods, cell type, organism, and DNA repair mechanisms. Continued research and technological advancements are expected to further enhance the precision, efficiency, and applicability of CRISPR-Cas9 for genomic editing in diploid organisms.

Please feel free to reach out if you have further questions or require additional insights on this topic.

Warm regards,

Take a look at this protocol list; it could assist in understanding and solving the problem.