CRISPR Technology for Genome Editing
Generally speaking, GOF(Gain of function) and LOF(Loss of function) validation are genetics methods to deeply study a gene’s function, which is necessarily required to publish a high-quality paper. GOF validation was achieved by overexpression of a gene through transgenic technology years ago by transient transfection or lentivirus infection with plasmids of strong promoter, while LOF validation was achieved by knockdown a gene’s expression with RNAi technology. Since 2013 when Feng Zhang have published a paper of CRISPR technology, more and more researchers turn to use CRISPR technology, which can do GOF via knockin as well as LOF via knockout. Moreover, the technology has been modified and can regulate gene expression (CRISPRa and CRISPRi).
(Ding et al., 2016)
The gene editing CRISPR technology has become the hottest tool and commonly used in molecular and cell biology because of its convenient manipulation and easy cost control. Here, we will discuss the technology from the perspective of the most commonly used gene editing stable cell line.
(Van Erp et al., 2015)
The workflow of gene editing CRISPR stable cell line construction mainly includes five steps:
Cell characterization & testing are often overlooked but it is very critical to the subsequent experiments. A good start is equal to half the success.
| Plasmid | Lentivirus | RNP (sgRNA+Cas9 protein) | |
|---|---|---|---|
| Cost | Low | Medium | High |
| Timeline | Medium | Long | Short |
| Cell types | Cancer cell lines | Cancer, primary and stem (iPSC) cells | Cancer, primary and stem (iPSC) cells |
| Species | Any species with appropriate promoters | Mammalian cells | Any species |
| Toxicity | Medium | High | Low |
| Cleavage efficiency | Low | Medium | High |
| Off-target effect | Medium | High | Low |
| Cas9 integration | Low efficiency | High efficiency | No integration |
| Application | KO/KI cell line engineering | KO cell line engineering | KO/KI cell line engineering |
If your cell line is commonly used in laboratories (such as HEK293, HeLa, and HCT116) or a cell line that is easily transfected after optimization (such as Jurkat), transfection with a plasmid is a fast and low-cost method. The commonly used plasmids are PX458 and PX459, both of which have been validated to work effectively in most cell types.
The screening marker for PX458 is EGFP, which can be used to enrich the transfected cells with FACS sorter after transfection. According to rule of thumb, rate of gRNA/Cas9 integration into the genome is low while the cutting efficiency is improved.
Figure 1. CRISPR knockout with PX458 (GenScript)
If you use resistance selection often, PX459 is a good choice. The screening marker for PX459 is Puro selection. In this case, the rate of gRNA/Cas9 integration into the genome was higher than that of PX458, which may resulting in higher off-target effect.
Particularly, Feng Zhang’s lab also released an enhanced version of the Cas9 plasmid, which is comparable in efficiency to the wild-type Cas9 plasmid, but the efficiency of off-targeting is much lower. In this case, enhanced version of PX459 plasmid is recommended. Enhanced plasmids (PX458-eSpCas9 and PX459-eSpCas9) are available for sale in GenScript.
| Service | Vector | Cas9 | Delivery | Vector Type | Selection Marker |
|---|---|---|---|---|---|
| eSpCas9 Plasmids | eSpCas9-2A- GFP(PX458) New | Enhanced | plasmid transfection | All-in-one Vector | EGFP |
| eSpCas9 Plasmids | eSpCas9-2A- Puro(PX459) V2.0 New | plasmid transfection | PuroR | ||
| eSpCas9 Plasmids | eSpCas9-LentiCRISPR v2 New | lentivirus | PuroR |
For difficult-to-transfect cancer cells, iPSCs or primary cells, lentiviral infection is usually used. Since lentivirus integrates the gRNA/Cas9 expression cassette into the genome, the off-target efficiency is higher than that of the plasmid transfection. In this case, it is strongly recommended to use the enhanced version of the gRNA/Cas9 plasmid. The enhanced lentiviral plasmid (eSpCas9-LentiCRISPR v2) is available for sale in GenScript as well.
Figure 2. CRISPR knockout in N2A cells with Lentiviral infection (GenScript)
For some cell types that are susceptible to virus infection, DNA transfection or off-targeting effect is concerned, RNP transfection is a good choice. The advantages of RNP are low cytotoxicity and low off-target efficiency. RNP can be delivered into cells by chemical transfection or electroporation, which is same as plasmid transfection.
In particular, when performing knockin experiments in difficult-to-transfect cells, co-transfection of gRNA/Cas9 plasmid and homologous donor template DNA is very toxic to cells. At this time, using RNP can lower the cytotoxicity, which is beneficial to subsequent monoclonal growth. It is reported the efficiency of knockin can be improved due to RNP’s higher cleavage efficiency.
Figure 3. CRISPR knockout in THP-1 cells with RNP
