CRISPR Point Mutation Stable Cell Line
VectorBuilder can engineer stable cell lines harboring desired point mutations at genomic target sites of interest. These point mutations are generated using a CRISPR-based approach wherein a target site specific gRNA/Cas9 RNP complex is introduced into the target cells along with a donor vector carrying the point mutation template for homology-directed repair (HDR). Our proprietary technology allows us to achieve very efficient gene delivery and HDR, resulting in rapid turnaround and high successful rate for obtaining homozygous mutants.
Highlights
- Supreme point mutation efficiency: cells exhibit HDR rate of up to 50% with our proprietary donor design and gene delivery approach.
- Non-viral delivery approach: electroporation-based RNP delivery is efficient and has minimal off-target effects.
- Rapid turnaround: homozygous mutant cells can be delivered in as fast as 18 weeks starting from vector design.
Service Details
Figure 1. Typical workflow of CRISPR point mutation stable cell line production.
Price and turnaround
| Service Type | Deliverable | Price (USD)* | Turnaround |
|---|---|---|---|
| Point mutation | One homozygous single clone (>106 cells/vial, 2 vials) | From $7,999 | 12-18 weeks |
* Additional charge will apply for extra single clones or vials.
QC assays
| Assay | Methods |
|---|---|
| Point mutation validation (default) | PCR, Sanger sequencing |
| Expression test (add-on) | RT-qPCR, WB, IF, FACS |
| Off-target analysis (add-on) | NGS, PCR, Sanger sequencing |
| Chromosome analysis (add-on) | Karyotyping |
| Sterility (default) | PCR for mycoplasma detection, bioburden test for sterility detection |
Downstream services
We can perform various phenotypic assessments and functional validation of your engineered cell lines, including assays for proliferation, apoptosis, migration, viability, cytotoxicity, etc.
Case Studies
Figure 2. Point mutation rates in various cell types using our optimized mutagenesis strategy.
Figure 3. Validation of point mutations in THP-1 cells. (A) Point mutations in the GOI were verified by Sanger sequencing. The red square indicates nonsynonymous mutations and the green square indicates synonymous mutations. (B) Lengths of the PCR product of target site are the same for cells with point mutation (PM) and wildtype (WT) cells, indicating no structural mutation in PM cells.
How to Order
Resources
FAQ
For smaller fragments containing the mutation to be introduced, ssODN is a good option because of technical simplicity and its inability to integrate at random locations in the genome. However, for ssODN the template must be less than 200 bp. For larger fragments, dsDNA donors can be used. In either case, efficiency is increased when the cut-to-mutation distance is minimized to 10 base pairs for ssODN and 1 kb for dsDNA donors.
Although synonymous mutations produce the same amino acid sequence, they can affect codon biases, mRNA secondary structure and stability, and protein translation rates. Introducing synonymous mutations in cell lines allows the study of the effects of these post-transcriptional changes. Additionally, introducing a synonymous mutation that disrupts the target sequence for CRISPR components (e.g. PAM and nearby sequences) can suppress unwanted cleavage at off-target and previously CRISPR-Cas9 edited site.
Featured Citations
The KU-PARP14 axis differentially regulates DNA resection at stalled replication forks by MRE11 and EXO1
Ashna Dhoonmoon, Claudia M. Nicolae & George-Lucian Moldovan.
Nature Communications, 2022, doi: 10.1038/s41467-022-32756-5
Products used:
Abstract: Suppression of nascent DNA degradation has emerged as an essential role of the BRCA pathway in genome protection.In BRCA-deficient cells, the MRE11 nuclease is responsible for both resection of reversed replication forks, and accumulation of single stranded DNA gaps behind forks. Here, we show that the mono-ADP-ribosyltransferase PARP14 is a critical co-factor of MRE11. PARP14 is recruited to nascent DNA upon replication stress in BRCA-deficient cells, and through its catalytic activity, mediates the engagement of MRE11. Loss or inhibition of PARP14 suppresses MRE11-mediated fork degradation and gap accumulation, and promotes genome stability and chemoresistance of BRCA-deficient cells. Moreover, we show that the KU complex binds reversed forks and protects them against EXO1-catalyzed degradation. KU recruits the PARP14-MRE11 complex, which initiates partial resection to release KU and allow long-range resection by EXO1. Our work identifies a multistep process of nascent DNA processing at stalled replication forks in BRCA-deficient cells.
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A lysine-rich cluster in the N-BAR domain of ARF GTPase-activating protein ASAP1 is necessary for binding and bundling actin filaments
Anjelika Gasilina, et al.
Journal of Biological Chemistry, 2022, doi: 10.1016/j.jbc.2022.101700
Products used:
Abstract: Actin filament maintenance is critical for both normal cell homeostasis and events associated with malignant transformation. The ADP-ribosylation factor GTPase-activating protein ASAP1 regulates the dynamics of filamentous actin_x0002_based structures, including stress fibers, focal adhesions, and circular dorsal ruffles. Here, we have examined the molecular basis for ASAP1 association with actin. Using a combination of structural modeling, mutagenesis, and in vitro and cell-based assays, we identify a putative-binding interface between the N-Bin-Amphiphysin-Rvs (BAR) domain of ASAP1 and actin filaments. We found that neutralization of charges and charge reversal at positions 75, 76, and 79 of ASAP1 reduced the binding of ASAP1 BAR-pleckstrin homology tandem to actin filaments and abrogated actin bundle formation in vitro. In addition, overexpression of actin-binding defective ASAP1 BAR-pleckstrin homology [K75, K76, K79] mutants prevented cellular actin remodeling in U2OS cells. Exogenous expression of [K75E, K76E, K79E] mutant of full-length ASAP1 did not rescue the reduction of cellular actin fibers consequent to knockdown of endogenous ASAP1. Taken together, our results support the hypothesis that the lysine-rich cluster in the N-BAR domain of ASAP1 is important for regulating actin filament organization.
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