How to plan my CRISPR experiment
How do you import Cas9 and gRNA?
In fact, the way you import cells is already determined as long as you choose to express the system.
However, it is still necessary to optimize the operation when the gRNA and Cas9 are imported into the cell, because the transfection efficiency changes with the import method and the cell type.
1, liposome transfection method: transfection DNA plasmid and the efficiency is higher, but Cas9 - gRNA ribonucleoprotein complex, due to the large protein, transfection efficiency will be low, need to choose high transfection efficiency of transfection reagents;
2, electroporation method: whether Cas9 / gRNA plasmid or Cas9 - gRNA ribonucleoprotein complex, its import efficiency were significantly higher than the liposome transfection, need DianZhuan instrument, and need to support the use of the instrument of reagent groped DianZhuan conditions according to different cell types, the construction cost is relatively high;
3, virus packing: adenovirus packing is suitable for almost all cell types, preparation, easy and simple operation, independent outside of the virus in the host genome replication, not integrated into the host genome, efficiency of infection is higher, but such as slow virus, a retrovirus is integrated into the genome of risk, the method is limited to import the nucleic acid, Cas9 - gRNA ribonucleoprotein complex can not use this method;
4. Microinjection: the transfer of foreign genes with microinjection, without limitation of length, has demonstrated that hundreds of KB of exogenous DNA fragments can be successfully transferred into cells.
Also can do precise into foreign proteins, and its transfer efficiency is much higher than liposome and DianZhuan way, very suitable for Cas9 - gRNA ribonucleoprotein complex method based on gene editing editing animal eggs.
The disadvantage is that the equipment is sophisticated and expensive, and it requires long hours of practice, and only a limited number of cells can be injected at a time.
2. How to verify genetic modification?
Now is the time for acceptance.
Some of your cells may be mutated cells, and some of them are wild cells.
This is because some cells lack the expression of gRNA or Cas9, or have no effective cutting after expression.
The modified cells may be homozygous and may be heterozygous.
In addition, non-homologous terminal connections (NHEJ) are prone to error, so each mutation's allele may be different.
Even though homologous guidance is repaired, a large number of double-stranded faults are still completed by non-homologous terminal connections.
Therefore, this heterogeneous group of cells will contain a variety of mutations and edits.
How to do?
You need to verify.
1.Mismatch cleavage method (applicable to NHEJ) : semi - quantification of the percentage of mutated alleles in mixed cells.
The area of interest was amplified by PCR, then the product was modified to be processed by the nucleic acid enzyme with the DNA of the cutting DNA, and then the gels were used to identify the DNA fragments.
2.PCR and restriction enzymes (for HDR) : suitable for small range nucleotide editing with novel restriction enzyme cutting sites.
The areas of interest were amplified by PCR, digested with appropriate restriction enzymes, and then the gels identified DNA fragments.
3.PCR amplification and gel electrophoresis (for HDR or NHEJ) : suitable for large insertion or deletion.
The PCR amplification was performed using the primers on either side of the target area or across the boundary of the inserted fragments.
Electrophoresis analysis of PCR products to determine whether the editor was successful.
4.PCR amplification, cloning and sequencing (for HDR or NHEJ) : semi-quantitative assessment of the editing effect and the exact sequence of alleles.
The area of interest was amplified by PCR, cloned into plasmid, and then screened single clones and sequenced.
At this point, the work of genome-editing is done.
Of course, our guide just provides a framework for your reference.
The CRISPR/Cas9 technology is growing so fast that new methods and applications are emerging.
Everyone must keep learning to keep up with the rapid pace of development.
