Exclusive CRISPR-U™ technology and stem cell culture system facilitate iPSC gene editing

Exclusive CRISPR-U™ technology and stem cell culture system facilitate iPSC gene editing

In August, Ubigene introduced the relevant CRISPR/Cas9 gene editing applications of iPSC in disease modeling, drug screening and gene therapy (Figure 1). Those who have not our previous atricle please click here >>What happened when iPSC met CRISPR?). After the article released, we received many inquiries, such as cell culturing, single-cell cloning, low positive rate, etc...To facilitate your iPSC gene editing experiments, we sorted out the issues that may happened and the relevant solutions. Hope this article could help!

Figure.1 Clinical applications of iPSC gene editing

Issues of iPSC culturing

1）Poor cell viability during culturing and easy to differentiate

Unlike tumor cells, iPSCs are more delicate. Here are some tricks to maintain cell viability and stemness of iPSCs. The basic aspect is the selection of culture medium and matrix. High-quality reagents must be selected to ensure adequate nutrition and a comfortable growth environment. The culture system used after thawing is recommended to be consistent with that before freezing. If the replacement of culture medium or matrix is necessary, it can only be replaced during passage, and cells would need some time to adapt. Moreover, unlike other tumor cells that can refresh culture medium once every two or three days, iPSCs need to refresh culture medium every day and subculture in time to avoid affecting cell activity and causing differentiation due to overgrowth or contact inhibition of colonies. During subculture, it should also be noted that cells cannot be over digested. In addition, the culture of iPSCs needs constant attention and care, including but not limited to observing iPSCs under phase contrast microscope (4x, 10x, 20x and 40x magnification) every day, and monitoring iPSC colony morphology, differentiation and confluence (Figure 2). The ideal colony of iPSC morphology should be compact inside, uniform in size and clear in edge. If there are a few differentiated cells at the edge, timely and adequately subculture should be made to maintain the pluripotency and logarithmic growth of iPSC.

Figure 2. iPSC colony ranking and differentiation levels (Source: sigmaaldrich)

2）Difficult to isolate single-cell clones

  Colony growth and expansion is the key stage to obtain positive gene-editing cells. This process is time-consuming and laborious. If the rate of colony formation is low, the workload will increase. The cell condition before cloning is very important. The better the cell condition is, the higher the rate of colony formation is. In addition, iPSC is easy to apoptosis after being digested into a single cell, and apoptosis inhibitor must be added to make the clones grow better.

Low gene editing efficiency in iPSCs

1）Strategy design and transfection efficiency

      The targeting strategy design is crucial to the success of the iPSC gene-editing project. For the knockout project, the selection of knockout size, the specificity and the cutting efficiency of sgRNA could affect the chance of getting positive cells. For point mutation or knockin project, the distance between the cutting position and the mutation locus or knockin position should be comprehensively considered. Meanwhile, the introduction of synonymous mutation at PAM position and the design of the donor homologous arm should also be taken into account. A good editing strategy is the basis for the successful implementation of the project. In addition, iPSCs from different individuals and tissues are quite different, and the transfection methods and parameters may be different. It is necessary to conduct preliminary experiments for each iPSC, to optimize the transfection parameters and improve the transfection rate. The improvement of transfection can greatly increase the success of gene editing.

2）HDR efficiency

      Compared with gene knockout, the project difficulty of point mutation and knockin in iPSC is higher. The main reason is that compared with immortalized cell lines, the efficiency of directed homologous recombination repair (HDR) in iPSCs is low ^[1]. HDR is the main method to repair CRISPR induced double strand breaks (DSB) by exogenous donor DNA. In order to overcome the low HDR efficiency, researchers have adopted several strategies, such as adding resistance genes to CRISPR plasmids or donor DNA. Although this method is effective, it will insert foreign DNA into the genome ^[2]; Using lox franked selection markers or PiggyBAC transposon system can improve HDR efficiency, but its construction cycle is also significantly prolonged ^[3]; Although the method of single-strand oligonucleotide (ssODN) donor avoids the problem of random integration of large double strand DNA molecules, it still suffers from the problems of low success rate of repair and sequence disorder around DSB sites ^[4]; There are also methods such as using known cell cycle inhibitors to synchronize cell cycle to achieve the purpose of timing delivery of Cas9 RNP complex ^[5], and using small molecule compounds to inhibit non homologous end direct connection (NHEJ). Among them, the addition of NHEJ inhibitor is more convenient and efficient, which is a common improvement method.

3）Genotyping

  As mentioned earlier, the workload of iPSC colony isolation is large. Before isolating single cells, strict quality control should be carried out on the transfected cell pool to ensure that the editing on the cell pool level is effective. Ubigene’s genotype analysis system (GAS) can be used for analyzing transfected cell pool genotype, confirming editing efficiency, guiding whether to proceed next step and the number of single cells should be isolated, achieving high-throughput interpretation of monoclonals genotype, screening positive clones, and saving a lot of labor. Click here to get a free trial>>

Case study

      Using RNP method to achieve c.G2149T point mutation in iPSC. After transfection, the recombination efficiency of the cell pool was detected. According to the Sanger sequencing result, gRNA had significant cutting efficiency (Figure. 3). According to the EZ-editor^TM GAS, homologous recombination genotype accounted for 14% (Figure. 4), which was high in efficiency. Single-cell clone isolation could be performed.

Figure 3. Sanger sequencing result of transfected cell pool

Figure 4. EZ editor^TM GAS result of HDR analysis

The issues and solutions are sorted out for you. You could now practice with confidence!

If you still hesitate to do it yourself, Ubigene provides customized services for iPSC knockout, knockin and point mutation. Our experience in iPSC gene editing ensures the delivery of high-quality gene-edited iPSCs. We have optimized stem cell media to easily improve iPSCs culture, tested at least three transfection methods for different iPSCs to ensure transfection efficiency, and invented unique EZ-editor^TM monoclone validation technology to high-throughput screen for positive clones. Click to learn more details of iPSC gene editing services>>

Related products:

Monoclone Validation Kit>>Allow early colony validation during monoclonal growth. 30-5 cells can get enough templates for PCR, only 15 minutes, amplify sequence as long as 10kb!

References:

[1] Hockemeyer D, Jaenisch R. Induced pluripotent stem cells meet genome editing[J]. Cell stem cell, 2016, 18(5): 573-586.

[2] Zhang Y, Schmid B, Nielsen T T, et al. Generation of a human induced pluripotent stem cell line via CRISPR-Cas9 mediated integration of a site-specific homozygous mutation in CHMP2B[J]. Stem cell research, 2016, 17(1): 151-153.

[3] Yusa K, Rashid S T, Strick-Marchand H, et al. Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells[J]. Nature, 2011, 478(7369): 391-394.

[4] Yang L, Guell M, Byrne S, et al. Optimization of scarless human stem cell genome editing[J]. Nucleic acids research, 2013, 41(19): 9049-9061.

[5] Lin S, Staahl B T, Alla R K, et al. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery[J]. elife, 2014, 3: e04766.