CRISPR Cell Lines
VectorBuilder offers a diverse range of single clone-derived and functionally validated cell lines expressing Cas9 or its variants. These CRISPR cell lines serve as flexible and powerful tools for gene knockout, knockin, activation, and inhibition, as well as high-throughput CRISPR screening, ensuring reliable results in your experiments.
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Shipping and storage
Cells are shipped on dry ice (in 10% DMSO-containing culture medium). Upon receipt, please immediately transfer the cryovials to the liquid nitrogen vapor phase for long-term storage. You can also store the cells at -80°C for short-term storage (≤1 week).
Note: For any premade stable cell lines purchased from VectorBuilder, customers agree not to resell them in any form, including in their original form, as copies, as aliquots, or in repackaged form, to any third party, unless stipulated differently in a separate agreement with VectorBuilder.
Technical Information
Figure 1. Simplified workflow for using VectorBuilder’s hCas9-expressing stable cell lines.
We offer a variety of CRISPR cell lines suitable for a wide range of applications. In cell lines stably expressing hCas9, knockout or knockin of the target gene can be achieved by introducing one or more gRNAs and, when applicable, a repair donor DNA template (ssODN, circular or linear dsDNA). Targeted gene transcriptional activation or repression can be achieved by introducing msgRNAs or gRNAs into cell lines stably expressing dCas9-SAM or dCas9-KRAB-MeCP2, respectively, enabling regulation of target gene expression without permanent modification to the genome. Additionally, our premade CRISPR cell lines are well-suited for high-throughput screening when compatible guide RNA libraries are introduced.
For more detailed information, please refer to our CRISPR Genome Editing Solutions and User Instructions for CRISPR System. To design guide RNA expression constructs or donor vectors for use with our premade CRISPR cell lines, please visit our Vector Design Studio or contact us for assistance.
Experimental Validation
Figure 2. Validation of hCas9-expressing K562 cells. Cells were transfected with a plasmid expressing a single gRNA targeting the hB2M gene. 72 hours post-transfection, genomic DNA was extracted, and the target region was amplified for Sanger sequencing. Indel decomposition analysis was then performed to assess editing efficiency (43.2% of sequence with mutation inferred by trace decomposition).
Resources
FAQ
Are the premade stable cell lines derived from mixed pools or single clones?
All of VectorBuilder’s premade stable cell lines are derived from single clones to ensure consistency and reproducibility. Our single clone stable cell lines undergo stringent QC and functional validation to provide you with full confidence in downstream applications.
Featured Citations
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.
J Biol Chem, 2022, doi: 10.1016/j.jbc.2022.101700
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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-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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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
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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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