Achieving stable expression of a specific phenotype within cells can be accomplished through point mutations, where specific genomic sites are replaced with foreign mutation sites through homologous recombination or homology-directed repair (HDR) pathways. However, in the actual research and development process, results often fall short of expectations. The development of cell lines comes with a myriad of challenges, from complications in obtaining knock-in positive clones and point mutation homozygotes, to problems with the cultured cells exhibiting poor health. Compared to gene knockout (KO) cell lines, the experimental design plans for point mutation cell lines require consideration of various factors, such as the length of gene fragments and homologous recombination efficiency. Different cell lines can exhibit significant differences in editing and homologous recombination efficiency, making their development more challenging. This is especially true for many human-derived stem cell lines and induced pluripotent stem cell (iPSCs) used in current research to model complex human diseases.
After extensive testing and process optimization, Cyagen has developed the Smart-CRISPR™ Cell Gene Editing System, enabling more in-depth gene information analysis and more efficient gRNA design. With this gene editing system, we can offer accurate and rapid construction services for point mutation cell lines. Using Cyagen's optimized α-donor system, we transfect humanized Cas protein, gRNA, and donor components into target cells, facilitating highly efficient homologous recombination — increasing HDR efficiency by over 660% compared to a traditional donor system for iPS cell pools (figure below) — and achieving homozygous model delivery.
|Industry Technical Challenges||Cyagen’s Solutions|
|Poorly designed gRNA and donor sequences result in low homologous recombination efficiency.||▶||
Our innovative Smart-CRISPRTM Cell Gene Editing System allows for the scientific design of high-efficiency, low off-target risk gRNAs, achieving editing efficiencies of up to 90%.
Our proprietary α-donor system boasts an HDR efficiency of up to 50%, significantly surpassing the editing efficiency of traditional donors on the market while also seamlessly enabling footprint-free repair.
|Inadequate cell transfection methods lead to low efficiency.||▶||
Various cell types, including tumor, non-tumor, stem cells, and iPSCs, can be adjusted to the logarithmic growth phase before transfection.
We support various transfection techniques, including calcium phosphate co-precipitation, artificial liposome method, electroporation, and viral infection, among others.
|Improper selection of delivery vectors causes significant cell death after transfection.||▶||The use of Ribonucleoprotein (RNP) delivery methods results in a cell viability rate as high as 90%, significantly enhancing gene editing efficiency in cell suspensions.|
|Monoclonal cell growth is time-consuming and cell preparation is challenging.||▶||Our unique preparation methods achieve a monoclonal formation rate of over 30% and we can obtain a sufficient number of positive clones with just one round of screening.|
|Disorganized monoclonal data.||▶||We can analyze point mutation efficiency and identify homozygous clones in as fast as 1 minute.|
|iPS cells (iPSCs) tend to differentiate and lose their pluripotent nature during the editing process.||▶||With 17 years of experience in stem cell projects, we can develop ideal iPSC colonies: internally compact, uniform in size, and with clear edges.|
|In vitro cell models have limitations and can exhibit significant discrepancies from clinical outcomes.||▶||Having point mutation mouse and rat models allows for better simulation of complex biological environments, facilitating the preclinical study of disease mechanisms and drug efficacy.|
|Type||General Cell Lines||Deliverables||QC||Turnaround||Order|
|Tumor immune cells||THP-1, Jurkat, HepG2, SK-MES-1 etc.||1 homozygous clones, 2 vials (1*10^6 cells/vial), genotyping report.||PCR + Sanger sequencing||As fast as 12 weeks|
|Non-cancer immortalized cells||HSF, AC16 etc.||1 homozygous clones, 2 vials (1*10^6 cells/vial), genotyping report.||PCR + Sanger sequencing||As fast as 12 weeks|
|Induced pluripotent stem cells||iPSCs||1 homozygous clones, 2 vials (1*10^6 cells/vial), genotyping report.||PCR + Sanger sequencing + immunofluorescence||As fast as 12 weeks|
|Stem cells||H1, H9||1 homozygous clones, 2 vials (1*10^6 cells/vial), genotyping report.||PCR + Sanger sequencing + immunofluorescence||As fast as 14 weeks|
|Gene Point Mutation (PM)||Cell Type||Unoptimized Traditional Donor System||Optimized α-donor System||Point Mutation Efficiency % Increase w/ α-donor|
|Gene 1 PM||HEK293||1%||13%||1200%|
|Gene 2 PM||iPSC||0%||27%||Enables PM!|
|Gene 3 PM||Hep-G2||2%||40%||1900%|
|Gene 4 PM||HK-2||2%||30%||1400%|
SCARB1 is the primary receptor for high-density lipoprotein (HDL), facilitating cholesterol uptake by the liver from HDL. Using CRISPR/Cas gene editing technology, liver cancer cells were modified to carry the SCARB1 (p.K500N, K508N) mutations. As shown in the figure, sgRNA and Donor sequences containing the point mutations were designed near the point mutations. The gRNA and Donor were delivered into Huh-7 cells using the α-donor system, resulting in a cell pool with a detected HDR efficiency of 40%. Monoclonal clones were prepared to obtain Huh-7/SCARB1 (p.K500N, K508N) biallelic point mutant homozygous clones.
Point mutation cell lines are used in various research applications to study the effects of specific genetic mutations on cellular processes and disease mechanisms. Here are the top 10 modern research applications of point mutation cell lines, in no particular order:
Cancer Research: Point mutation cell lines are essential for understanding the role of specific mutations in cancer development, progression, and drug resistance. They help in evaluating potential targeted therapies and drug screening.