If the cells are stably expressing the Cas9 and sgRNA, the momoallelic knockout becomes biallelic. You need to keep the culture in the selection medium (eg. puromycin) to prevent the growth of cells without stable expression of Cas9 and sgRNA. However, you need to understand that stable expression of Cas9 increases the chances of off target effects of the sgRNA, which may effect your experiments in a long run. Good luck

Dear Ramachandramouli Budida,

Thank you for the reply. Intuitively that makes sense, have you observed such phenomenon for any of your CRISPR/Cas9 knockouts?

Hello Ayon,

Cas9 will edit both alleles, but sometimes it just restores the original open reading frame by introducing a SNPs in the PAM that cannot be edited further. For these reasons having the Cas9 stably expressed it's not going to turn all your cells into homozygous KO. You could possibly observe a marginal enhancement but overall it is not recommended as off-targets will increase.

Hope it helps

Hi Francesco, thanks for the reply. I was also thinking that this could be a possible reason why monoallelic knockouts are so common. However, if the reason for one allele not being knocked out is indeed because the Cas9/sgRNA cannot induce a DSB anymore, I was wondering how the invitro validation assays using the same Cas9/sgRNA work? For instance, the in vitro cleavage assay from clonetech (see attached image) requires the wildtype sequence from a monoallelic KO line to be targeted in order to distinguish between hetero- and homozygous KO, meaning that somehow in the cells Cas9/sgRNA is not targeting the site anymore but it can do so in vitro. Any thoughts on this?

@Ayon

The Cells with stable Cas9 and sgRNA expression were acting weird in our lab. We find out that this is due to off target effects.

Hi Ayon, the screening you are mentioning should be done on PCR amplicons from single cells or clonal pools (amplified from a single cell) after transfection with Cas9 and sgRNAs. Cas9 is very efficient but you can only have a readout when it introduces an indel that inactivates further cuts. Most of the time instead, the DBS is just repaired without mutations. Even when a mutation occurs you will instead produce a frameshift 2 out of 3 times and depending on the locus the probability of a meaningful KO can be even lower. The assay you are showing, providing that you are using single cells PCR makes perfect sense. And it will allow you to distinguish between homozygous and heterozygous pretty well. However some of the genetic homozygous mutations can still produce your protein on one allele, on account of some mutations that restores the ORF.

If you found that some of your clones with this assay (from single cells or clonal pools) are heterozygous using this assay, it means that the other allele is truly a wt and you should find better way to express Cas9 in your cells. The levels you are using might not be enough. Cas9 is always more efficient in vitro.

Hope it helps

Dear Francesco,

Thanks a lot for taking your time to write a detailed reply. Yes, I understand your point about the inefficiency of NHEJ to induce a KO mutation on both alleles.

I was just wondering why it is so common to obtain single clones with one monoallelic KO and one truly WT sequence, since these cells definitely have enough Cas9/sgRNA expression to induce DSB so the truly WT sequence should be targeted with time. So, biologically speaking, in a monoallelic KO single clone, should we not expect a non-KO mutation on the other allele to be more common than a truly WT sequence?

We are knocking out genes with CRISPR/CAS9 in plants, and we get monoallelic mutations frequently (to our great frustration). We find that CAS9 is integrated and is intact, but perhaps it or the guide RNA, or CAS9 is not getting expressed. I wonder if they may be getting silenced.