Fail to get KO homozygotes, the reason is that you may knock out the essential gene!
Even having rich experience in gene-editing, you may experience the following cases:
1) The transfection efficiency is high, observing a high coverage of green fluorescence;
2) gRNA cutting efficiency is high, double peaks at PAM site is observed from the sequencing of the pool;
But once you get the sequencing results of the single-cell clones, only WT cells or heterozygotes (KO+WT) is obtained, or with 3-n deletion. Then you run the experiment again, but still, the same result is obtained. Have you considered the reason for this scenario?
Knocking out the gene of interest from cells, and observing the resulting phenotype, is a common strategy used in biological research to determine gene function, such as for studying cellular signal transduction, regulation of gene expression, or structural composition. However, there are genes that are essential for cell viability and the knockout of these genes can cause cell death, and it is likely that the scenario described in the introduction is the results of knocking out an essential gene therefore unable to get KO homozygotes.
According to the results of genome-wide screening, essential genes account for approximately 10% of the total human genes . If you're not sure if a gene is an essential one for a cell, is there a way to search for it prior to the experiment? The first thought may be to look up the literature to see if there are successful knockout cases of the same genes and cells of interest. But after all, it is time and effort to review a bunch of articles, and often the exact same cases with the same gene and cell line cannot be found. With the wide application of CRISPR library screening, more and more cell genome-wide knockout data are disclosed. Ubigene has gathered nearly 30 million CRISPR library screening data from 1400 + cell types, allowing you to assess project feasibility (Figure 1&2) and find success cases (Figure 3) simply by entering cell and gene names. Try it for free >>>
In other words, if you still want to perform the knockout of a high-risk gene, is it no way to achieve it? Of course not, Ubigne has sorted out 3 major solutions for you based on years of experience with gene-editing cell construction.
1) Alternative method to knock down the gene. If the knockout of the gene leads to cell death, it is possible to observe whether there are phenotypic changes by repressing the expression of the gene by RNAi. If the knockdown has a phenotype, generally, the result has a certain value for your publication.
RNAi case study: downregulation of MTA1 expression by an RNAi approach can lead to ER α re-expressed in ER-negative breast cancer cell line MDA-MB-231, the decrease of the protein levels of MMP-9 and CyclinD1, as well as reduced tumor cell invasion and proliferation. More cells were blocked in G0/G1 phase (P < 0.05). The silencing effect of MTA1 could effectively inhibit the invasion and proliferation of MDA-MB-231 cells. shRNA interference against MTA1 may have potential therapeutic utility in human breast cancer .
RNAi literature case: down regulation of MTA1 expression by an RNAi approach can lead to ER α Re expressed in Er negative breast cancer cell line MDA-MB-231, and decreased the protein levels of MMP-9 and CyclinD1, as well as reduced tumor cell invasion and proliferation, more cells were blocked in G0 / G1 phase (P < 0.05). The silencing effect of MTA1 could effectively inhibit the invasion and proliferation of MDA-MB-231 cells. ShRNA interference against MTA1 may have potential therapeutic utility in human breast cancer .
2) Challenge the probability. If the interference effect is not good, but the gene is also just lethal with some probability, you can try isolating more clones to see if you can be fortunate to obtain homozygotes. If no homozygote is obtained but a KO heterozygote or KO pool, you can also detect the expression of the cells. Sometimes KO heterozygotes also have a phenotype that is available for publication.
KO heterozygotes case study: NAT10 is a nucleolar protein containing an acetyltransferase domain and a tRNA binding domain with histone acetylation activity. NAT10 is involved in the regulation of human telomerase reverse transcriptase. This gene is an essential gene in the vast majority of tumor cells and the knockout of this gene leads to cell death. One of the researchers used CRISPR technology to perform knockout in LoVo cells and the KO heterozygote (NAT10+/-) was obtained and performed with immunofluorescence, Western blotting, and sequencing validation. Compared to control cells (LoVo), NAT10+/− cells showed reduced MN formation, i.e. downregulation of NAT10 expression can inhibit colorectal cancer progression .
Figure 4. Downregulation of NAT10 expression reduced MN formation
Here is a supplementary tip, by adding some special supplements to the media to additionally replenish the nutrients needed for cell growth, the cells with the essential gene knocked out (that induces lethal) can grow and proliferate normally. Until the cells expand to sufficient amounts, the special supplements can be withdrawn and the cells can be used for functional validation experiments. For example, after the knockout of the DHODH in leukemia cell lines, the cells require ~100µM uridine added to the culture medium to maintain cell growth .
3) Adjust strategy to conditional knockout. As demonstrated by using the tet-off system in combination with CRISPR/Cas9. First to exogenously express the gene of interest using the tet-off system, followed by knockout of the endogenous gene. Then screen for KO homozygous clones, and after expansion to a sufficient amount, add dox to switch the expression of the exogenous gene off, achieving inducible gene knockout . Or by using an Auxin-inducible degron system (AID) in which AID is knocked in to form a fusion protein replacing endogenous gene expression, then induces the protein degradation by auxin addition, achieving gene function knockout . In addition, there are strategies to combine CRISPR/Cas9 and cre/loxp systems for conditional knockout.
Figure 5. Conditional knockout strategy 1
Figure 6. Conditional knockout strategy 2
The above 3 solutions, each with advantages and disadvantages. The 1st RNAi alternative method is the easiest to operate, less expensive, and has a high success rate, but there is a possibility that the interference effect is not good and the knockdown is not thorough. The second, the approach to increase the number of clones to be isolated and screen, is appropriate only for genes with lower lethality, has higher uncertainty, and needs more time and costs. The third, conditional knockout strategy is the most costly, the tet-off system may also be affected by background expression in some cases, and the KI approach is more difficult. Overall, the choice of strategies can be made by combining time, funding and specific genes.
Ubigene’s KO cell bank now is expanded to 3500 + KO cells, covering 1200+ genes from 26 signaling pathways, 1500+ genes from 27 disease types and 800+ genes from 5 major classes of drug targets, as low as $1980, as fast as 1 week to deliver, click to search > > > Ubigene also offer express customization, as fast as 4 weeks only. With comprehensive project evaluation and gene dependency assessment, Ubigene can help you achieve the experiment goal in one-stop and more reliably!
Wang T, Birsoy K, Hughes N W, et al. Identification and characterization of essential genes in the human genome[J]. Science, 2015, 350(6264): 1096-1101.
 Jiang Q, Zhang H, Zhang P. ShRNA-mediated gene silencing of MTA1 influenced on protein expression of ER alpha, MMP-9, CyclinD1 and invasiveness, proliferation in breast cancer cell lines MDA-MB-231 and MCF-7 in vitro[J]. Journal of Experimental & Clinical Cancer Research, 2011, 30(1): 1-11.
 Cao Y, Yao M, Wu Y, et al. N-acetyltransferase 10 promotes micronuclei formation to activate the senescence-associated secretory phenotype machinery in colorectal cancer cells[J]. Translational Oncology, 2020, 13(8): 100783.
 Sykes D B. The emergence of dihydroorotate dehydrogenase (DHODH) as a therapeutic target in acute myeloid leukemia[J]. Expert opinion on therapeutic targets, 2018, 22(11): 893-898.
 Liao J, Karnik R, Gu H, et al. Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells[J]. Nature genetics, 2015, 47(5): 469-478.
 Nishimura K, Fukagawa T. An efficient method to generate conditional knockout cell lines for essential genes by combination of auxin-inducible degron tag and CRISPR/Cas9[J]. Chromosome Research, 2017, 25: 253-260.