After deciding upon a CRISPR-mediated gene knockout strategy and obtaining the gRNA, the next step would be choosing the suitable strategy for cell transfection to introduce gRNA and Cas9 protein into cells. The efficiency of this transfection step directly affects the overall success rate of CRISPR/Cas-mediated gene editing and subsequent model generation.
Cell transfection methods include various approaches, which can provide either transient or stable expression in transfected cells. Common transfection methods and techniques include:
Transfected cells must express both gRNA and Cas9 protein in cells at the same time to achieve a gene KO. Therefore, no matter what method we choose, the transfection efficiency should be ensured, since low transfection efficiency will lead to the failure of obtaining homozygous knockout monoclonal cells.
1. Ribonucleoprotein (RNP) Delivery Transfection: A large amount of Cas9 protein and gRNA are directly transfected into cells by electroporation, and the target gene could be cleaved after the RNP complex is formed in the cell.
Given that there is no need to construct plasmids or viral packaging, the experimental process of RNP method is the simplest and the whole experimental cycle is relatively short. Generally, the cell gene editing process can be completed in 5-6 days after transfection. Moreover, RNP method provides low off-target probability, no risk of DNA integration, and promoter compatibility - it is considered more efficient compared with the conventional plasmid-based gene editing method.
2. Viral Delivery Transfection: Lentivirus (LV) knockout plasmids are constructed, packaged, purified, and then transfected into cells, so as to cut the target gene, integrate the expression elements of gRNA and cas9 protein into the genome, and stably express gRNA and cas9 protein.
Because of lentivirus’ excellent infection capabilities in cells, the knockout efficiency with this method is high. However, it also has its limitations, such as higher cost. Virus packaging is time-consuming, and the random integration of lentivirus into the genome may affect other genes. Moreover, virus transfection is not suitable for suspension cells or virus sensitive cells.
3. Conventional Plasmid-based Method: The conventional knockout plasmids are constructed and transferred into cells by electroporation, transiently expressing Cas9 and gRNA in cells, to cut the targeted gene.
The experimental procedure of conventional plasmid-based transfection is relatively simple with short turnaround time and low cost, but its transfection efficiency is low.
Compared with RNP method, this method has a process to achieve expression, which further reduces its efficiency.
4. PiggyBac (PB) Transposon Method: The plasmids carrying the PB transposon and PB transposase (PBase) are cotransfected into the cells. PB transposase will identify the palindrome sequence on the transposon, and randomly integrate high-copy Cas9 protein and gRNA expression elements into the genome, so as to achieve the stable expression of gRNA and Cas9 protein.
Each of the four methods has its pros and cons. The best transfection method should be decided based on systematic analysis of the target genes and cells.
We can perform a variety of knockout (KO) strategies to generate a custom cell line model, including frameshift mutation, large fragment knockout, and multiple genes knockout. We will adopt the best knockout strategy to greatly improve the success rate of target gene knockout and the expression efficiency according to each project's unique requirements.