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  4. Point Mutation Cell Lines As Low As $8,500: Homozygous Delivery, As Fast As 12 Weeks

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.

Figure 1: Point Mutation (PM) HDR Efficiency of Traditional Donor versus Cyagen's optimized α-donor system, with % values generated by the HDR analysis software. For the PM of Gene 1, the optimized α-donor system demonstrates an HDR efficiency of 13%, 37%, and 46% compared to the traditional donor system HDR efficiency of 1%, 3%, and 6%, for HEK293, NCI-H1299, and iPSCs, respectively. For the  PM of Gene 2, the optimized α-donor system demonstrates an HDR efficiency of 27% compared to the traditional donor system HDR efficiency of 0% for iPSCs. For the  PM of Gene 3, the optimized α-donor system demonstrates an HDR efficiency of 40% compared to the traditional donor system HDR efficiency of 2% for Hep-G2. For the  PM of Gene 4, the optimized α-donor system demonstrates an HDR efficiency of 30% compared to the traditional donor system HDR efficiency of 2% for HK-2 cells. Table 1 presents the data alongside the increased PM efficiency that may be provided by our optimized α-donor system.
Cyagen’s α-Donor strategy significantly enhances knockin (KI) efficiency to develop point mutation cell lines, including iPSCs, with seamless integration and minimal off-target effects in as fast as 8 weeks. When you place your order now, you can enjoy a discounted price of $8,500 for point mutation projects in various cell lines such as A549, BEAS-2B, HCT116, HEK293, HeLa, iPSCs, and more.
Technical Workflow
AI-Enhanced Rare Disease Data Center (RDDC) Empowering Translational Medicine
With hundreds to thousands of mutation sites, how can you quickly pinpoint your target? By using a combination of gene diagnostic tools such as RNA splicing prediction, mutation pathogenicity prediction, mouse phenotype prediction, ASO prediction, SNP visualization, and more, you can save time and effort in your rare disease research.
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Challenges and Solutions in Point Mutation Cell Line Technology
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.
Point Mutation Cell Line Services
Special Offer on Select Point Mutation Cell Lines
Cell HEK293T A549 HCT 116 HEK293 Hela BEAS-2B HK-2 iPSCs
Order
Duration: Offer ends December 31, 2023
Promotion Details: Point mutation projects for the above cell lines and iPSCs can achieve homozygous delivery in as fast as 12 weeks and enjoy a discounted price of $8,500.
Available Cell Lines: Over 200+ Successful Cases
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
Note: In addition to point mutations, our platform also offers a wide range of gene editing cell line construction services, including gene knockout, knock-in, gene overexpression, stable interference strains, and more. Contact us now for more information!
Point Mutation Cell Line Service Case Studies
01.  Point Mutation Rates in Various Cell Types Using Cyagen Optimized Mutagenesis Strategy
02. SCARB1 (p.K500N, K508N) Biallelic Point Mutations
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%
NCI-H1299 3% 37% 1133%
iPSC 6% 46% 667%
Gene 2 PM iPSC 0% 27% Enables PM!
Gene 3 PM Hep-G2 2% 40% 1900%
Gene 4 PM HK-2 2% 30% 1400%
Table 1: Data used in Figure 1 (above). The point mutation efficiency of the target gene in each mixed cell pool is calculated by analysis software. At the cell pool stage, there are heterozygous, homozygous, and wildtype (WT) clones, but the distribution of heterozygous versus homozygous point mutations of the gene remains unclear at this stage. However, with higher cell pool point mutation efficiency,  the greater the opportunity to obtain homozygous clones.

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.

Advantages of In Vivo and In Vitro Gene Editing Platforms
A Mature Technical Platform
Cyagen has over a decade of experience successfully developing tens of thousands of gene editing projects across in vivo models (mice/rats) and in vitro models (cell lines/iPSCs/etc.). With over 1500 successful point mutation cell line cases and multiple published citations from scientific journals, we offer comprehensive services that span from cell gene expression regulation and functional validation to the construction of murine disease models and phenotype analysis, encompassing both in vivo mouse and rat research alongside in vitro cell studies.
Innovative Smart-CRISPR™ Technology
Our cutting-edge cell gene editing system, Smart-CRISPR™, effortlessly enables various strategies like gene knockout (KO), knock-in (KI), and more, with editing efficiencies reaching as high as 90%.
Large-Scale and Comprehensive Research Cell Repository
We have built a scientific research cell bank with clear parameters and stable performance, which has greatly avoided the negative impact of gene editing on cells. Currently, we have more than 300 types of target cells, more than 1,500 successful cases of cell lines, and multiple reference citations published across scientific journals.
Stringent Quality Control System
We employ a rigorous quality control system that includes dual testing for bacteria, mycoplasma, and other microorganisms to ensure 100% contamination-free products according to our specific pathogen-free (SPF) protocol. We also conduct routine thorough assessments of gene editing outcomes and perform cell viability checks to guarantee quality of deliverables.
Professional Project Management
We reply to all inquiries within 48 hours to provide prompt proposal development & quotes. All projects come with monthly progress updates, detailed delivery reports, and technical support from our team of experts. We also offer the flexibility to provide different clones (homozygous, heterozygous, and control) to meet your requirements.
Research Applications of Point Mutation Cell Lines

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.

1. Drug Discovery and Development: These cell lines are used to test the efficacy and safety of new drugs and potential therapeutic compounds for a range of diseases, especially when mutations are known to be involved.
2. Genetic Disease Studies: Point mutation cell lines enable the investigation of genetic diseases caused by specific mutations, providing insights into disease mechanisms and potential treatment strategies.
3. Functional Genomics: Researchers use these cell lines to explore the functional consequences of specific mutations on cellular processes and gene expression, aiding in the identification of potential therapeutic targets.
4. DNA Repair Mechanisms: Point mutation cell lines can be employed to study DNA repair pathways and their response to mutations, helping to elucidate the molecular mechanisms of genome maintenance.
5. Neurodegenerative Diseases: Researchers use point mutation cell lines to study mutations associated with neurodegenerative diseases like Alzheimer's and Parkinson's, allowing them to investigate disease mechanisms and develop potential treatments.
6. Rare Disease Research: For genetic-based rare disease research, point mutation cell lines help scientists model disease pathogenesis, screen for potential therapies, and gain a better understanding of the underlying biology.
7. Immunology and Autoimmune Diseases: These cell lines are used to investigate the impact of specific mutations on immune system function and their contribution to autoimmune diseases.
8. Stem Cell Research: Point mutation cell lines are used in the study of pluripotent stem cells & iPSCs to understand the effects of mutations on differentiation, development, and regenerative medicine applications.
9. Aging Research: Researchers use these cell lines to study the effects of specific mutations on cellular aging processes, helping to uncover insights into aging-related diseases and potential interventions.
These applications demonstrate the versatility and importance of point mutation (PM) cell lines in advancing our understanding of genetics, disease mechanisms, and the development of novel therapeutic strategies in preclinical research. PM cell lines are important models researching neurodegenerative and rare diseases, with their demonstrated use across many fields, including: neurology, immunology, aging, stem cell research, functional genomics, and more.
Next-Generation Humanized Genomic Ortholog Models: HUGO Mice
In addition to in vitro cell models, Cyagen is able to develop complex custom genome engineered mouse and rat models for research. Our next-generation Humanized Genomic Ortholog (HUGO) mouse models provide a platform with the highest degree of genetic humanization possible for studying genetic diseases, aiming to improve the translational outcomes for preclinical research. Additionally, we have established over 16,000 strains of knockout (KO) and conditional knockout (cKO) mouse models in our Knockout Catalog Models. Leveraging our in vitro/in vivo gene editing platform, we offer comprehensive services that encompass the entire research model process: from cell gene expression regulation and functional validation, to the development of mouse and rat models of disease, phenotype analysis, downstream breeding, and more. Contact us at 800-921-8930 or email animal-service@cyagen.com for more information.