SNP research needs point mutation cells - Summary of 4 construction methods | Ubigene

SNP research needs point mutation cells -

Summary of 4 construction methods | Ubigene

With the rapid development of NGS, more and more human single nucleotide polymorphisms (SNP) have been proved to be closely related to some human diseases. To study the pathogenesis or treatment of these SNP-related diseases, it needs to use the related point mutation cell models. However, it is not easy to construct point mutated cells. Choosing the right method can make you get twice the result with half the effort. What are the construction methods for point mutation cell lines? Let Ubigene share with you——

Most efficient method——Base editing

Principle description: The plasmids expressing gRNA, dCas9/nCas9, and deaminase will be co-transfected into cells. A single base of the gRNA target site will be replaced by deaminase without double-strand breaks. It has the ability for efficient and accurate gene-editing and can introduce point mutations in animal and plant cells, also correct pathogenic point mutations. Among them, adenine base editor (ABE) can enable A→G/T→C single base conversion for the target site, and cytosine base editing (CBE) can enable C→T/G→A single base conversion for the target site. At present, BE3 system (the third-generation base editor) is mainly used.

Application: It needs to be the transformation for specific bases and appropriate gRNAs can be designed. It fulfills the needs of disease treatment such as mutation correction.

Experimental workflow:

1. For the strategy design, gRNAs need to be found near the point mutation site, so that the target mutation site is within the editing window of the base editor, and there are no other non-target mutations of the same base in the editing window. There is no need to design the Donor.

2. The editing system will be introduced into cells by using plasmids (dsDNA).

3. Electroporation or liposome methods are used for cell transfection in most cases.

4. It is unable to screen positive clones only by PCR. Generally, sequence alignment (sequencing) will be needed.

Advantages: Gene-editing efficiency is quite high, and single base mutation can be accurately realized. It will not cause DNA double-strand breaks and reduce the occurrence of indel. It is safer and can reduce the off-target rate.

Disadvantages: It can only realize the conversion between specific single bases and cannot meet most scientific research needs. It cannot be used if there are other non-target mutations with the same base in the editing window. There are many restrictions on the application.

Most mainstream method - RNP method

Principle description: The gRNA and Cas9 protein form an RNP complex in vitro, which is then co-transfected with single-stranded oligo into cells, and the recognition and cleavage of genomic targets will be achieved by the RNP complex, followed by homologous recombination repair using single-stranded oligo as a template to achieve the purpose of gene point mutation.

Experimental workflow:

1. For the strategy design, gRNA with high specificity and cutting efficiency need to be found within 20bp upstream and downstream of the target mutation site. The single-stranded oligo template doesn’t have to be too long, a length of about 120nt is sufficient.

2. sgRNA and Cas9 protein will be treated as the vector.

3. Electroporation or liposome methods are used for cell transfection in most cases.

4. It is unable to screen positive clones only by PCR. Generally, sequence alignment (sequencing) will be needed.

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Advantages: Wide applicability, high editing efficiency, simple process, and short experiment turnaround.

Disadvantages: There are some spatial limitations associated with the need to select gRNAs near the target mutations; RNA is easily degraded and highly demanding for operation.

Breakthrough of the limitations of the RNP method - Plasmid resistance method

Principle description: The plasmid expressing gRNA and Cas9 together with the Donor plasmid will be co-transfected into cells. Within the Donor plasmid, a resistance gene expression cassette with LoxP recombination sites at both ends will be constructed between the two homology arms, and the target point mutations will be introduced by homology arms. After transfection, cells without successful recombination will not survive after antibiotic screening and the possibility of obtaining positive clones will increase. And after the positive clones are successfully screened, a plasmid expressing Cre recombinase will be transfected into the cells to delete the resistance gene then the cell model construction is completed.

Application: Applicable for some point mutations where the RNP method cannot design the gRNAs.

Experimental workflow:

1. For the strategy design, gRNA are usually designed at the intron near the mutation sites, and the left and right homology arms will be designed centered on the cutting site with a length of about 600-1000bp.

2. gRNA, Cas9 and Donor will be delivered via plasmids (single-stranded DNA).

3. Electroporation or liposome methods are used for cell transfection in most cases.

4. The positive clones can be preliminarily screened by PCR, then further confirm by sequencing (sequence alignment).

Advantages: Less limitation by the location of the target mutations; Cell screening by antibiotics can increase the possibility to obtain positive clones.

Disadvantages: Complicated process, long experimental turnaround; After the deletion of the resistance gene, LoxP sites remain at the intron.

Breakthrough method for difficult-to-transfect cell lines - AAV-Donor method

Principle description: Using AAV as a vector to deliver the Donor template for homologous repair, the AAV genome is free DNA single strand and can remain in the cell for a relatively long time, which can greatly improve homologous recombination efficiency.

Application: Applicable for some cell lines which have difficulty in transfections, especially the suspension cell lines.

Experimental workflow:

1. For the strategy design, gRNAs need to be found within 50bp upstream and downstream of the target mutation site, and the left and right homology arms will be designed centered on the cutting site with a length of about 600-800bp.

2. Generally, Cas9 will be stably expressed in the cell lines using lentivirus method, and sgRNA and Donor will be constructed on the AAV plasmid.

3. AAV method is mainly used for cell infection (delivering sgRNA and Donor) and lentivirus method can be used as the helper (delivering Cas9).

4. Generally, the AAV plasmid does not carry the resistance gene, so it is unable to screen positive clones only by PCR and sequence alignment (sequencing) will be needed.

Advantages: AAV is a single-stranded DNA virus, free in the cells as a template which enables high recombination efficiency. Applicable for difficult-to-transfect cell lines such as suspension cell lines. AAV has a relatively high safety profile in clinical applications, so in terms of disease therapy, the future is promising.

Disadvantages: Long turnaround and high cost for AAV packaging. Different cell lines need different serotypes for the AAV, so the feasible serotype needs to be tested with long turnaround and high cost.

Overall, Base editing method has the most efficiency and relatively easy process, but the threshold is also relatively high. If the design requirements of base editing method cannot be met, it is usually the case to choose the RNP method; If appropriate gRNAs near the mutation site cannot be found as per the strategy design principle of the RNP method, the plasmid resistance method can be considered to expand the selection range of gRNAs. For suspension cells that are difficult to transfect, the AAV method can also be chosen to address transfection problems and increase recombination efficiency.

So what if every researcher chooses the same system/method, will the chance to get the point mutated cell lines be the same for everyone? In fact, the whole experimental route can be optimized in many details, e.g. introducing synonymous mutations into the PAM structure can prevent re-cutting; Using the Cas9 stable expression cell line to construct point mutated cell lines can enhance transfection efficiency; Or adjusting the homology arm length and GC content, as well as adding reagents that inhibit the NHEJ repair pathway after transfection and incresing the homologous recombination efficiency can also help boost the point mutation efficiency. And to perform well optimization on these experimental phases, it requires continuous experiments and certain experience.

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