Cas9 Stable Cell Lines

Product DetailsApplicationCase StudyFAQ

Product Details

CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 (CRISPR associated protein 9) system is adapted from a naturally occurring bacterial immune system that can protect bacteria from damage caused by phage infection. As an unimagined tool for genome engineering, CRISPR/Cas9 is now commonly used. Compared with ZFNs and TALENs, CRISPR/Cas9 employs sgRNA (single guide RNA) to specify editing which makes the system more precise, time/cost-saving and quite valuable for high-throughput genome editing.

Creative Biogene has developed a series of Cas9 expressing stable cell lines. The Cas9 encoding gene is stably integrated into either random site, or AAVS1 safe harbor site of human genome, or ROSA26 safe harbor site of mouse genome. Apart from HEK293 and HeLa cells, we have achieved Cas9 stable expression in several types of cancer cells which makes it convenient for scientists to study related gene functions of certain cancers. For consistent, high-level and more stable expression of Cas9, monoclonal cells are isolated. The activity of Cas9 in each constructed stable cell line is functionally validated by the means of T7 Endonuclease I assay.

Key Features of Our Cas9 Stable Cell Lines:

Stable expression of Cas9 in several cell types including liver cancer cells, breast cancer cells, colon cancer cells, lung cancer cells, pancreatic cancer cells, gastric cancer cells, embryonic kidney cells etc.
Functional validation of Cas9 activity by T7 Endonuclease I assay.
Reduce the time required to determine gene function without generating your own Cas9 cell line.
Reduce the cost of a single experiment - no need to add nucleases.
Free of mycoplasma contamination.

Cas9 Stable Cell Lines Product List

Application

CRISPR/Cas9 system used for genome engineering in molecular biology is composed of two components: sgRNA which is a combine of original trRNA and crRNA, and Cas9 endonuclease. By delivering Cas9 and sgRNA into a cell, Cas9 is targeted to a given locus based on sequence complementary between sgRNA and cell genome, and further induces a double strand break (DSB). In the presence of donor template, the DSB can be repaired by HDR (homology-directed repair) pathway enabling precise editing such as point mutation. When DNA repair template is not provided, the cell is forced to undergo NHEJ (non-homologous end joining) pathway resulting in indels (insertions or deletions). Apart from wild-type Cas9, a few variants have been developed for reducing off-target effects or for other applications. SpCas9-HF1 is a high-fidelity variant designed to reduce non-specific DNA contacts.

Applications for Cas9 stable cell lines include:

Ideal cell models for downstream genome editing experiments including gene knockout, gene mutagenesis, gene knockin, and gene tagging: Cas9 stable cell lines make genome editing easier than ever. You simply deliver your target-specific gRNA into a CRISPR-ready cell line and select knockout or knockin cells for your desired target. There’s no need to make CRISPR-containing cas9 clones or worry about difficult-to-deliver Cas9 constructs. You only need to generate the target gRNA construct, which is easier to clone and deliver in a much smaller size.
sgRNA library screening: One of the most important applications of stable cell lines expressing Cas9 is in sgRNA library screening. CRISPR sgRNA libraries are collections of hundreds to thousands of plasmids, each expressing a unique sgRNA, and are useful for forward mutagenesis screens. CRISPR sgRNA libraries enable researchers to simultaneously knock out many genes in mammalian cells, opening the door to high-throughput drug target identification and validation, phenotypic changes, and reporter gene analysis.
sgRNA functional validation: Cell line-based CRISPR genome editing projects typically require several months of work, including transfection, clone isolation, and screening. While CRISPR generally provides a highly efficient method of genome editing, individual sgRNAs themselves do exhibit differences in efficiency due to the nature of individual target sites. It would be helpful to determine which CRISPR sgRNAs are most likely to be successful in genome editing before undertaking extensive screening efforts. Therefore, we can perform sgRNA functional verification in Cas9 stable cell lines.

Case Study

Case Study 1

Generation of knockout clones in mammalian somatic cell lines via stable cell lines expressing Cas9. When using a dual-vector system, a stable cell line expressing Cas9 must first be generated. This cell line can then be reused in multiple knockout experiments. Creative Biogene offers Cas9-expressing cell lines, some of which have inducible Cas9 expression.

Figure 1. Schematic outline of the knockout process. (Giuliano C J, et al., 2019)

Case Study 2

CRISPR screens take advantage of the efficiency and flexibility of CRISPR-Cas genome editing. They have become popular and efficient tools for biological discovery in a wide range of applications. In a typical hybrid CRISPR screen, a CRISPR guide RNA (gRNA) library is introduced into cells in batches so that individual cells receive different gRNAs, and perturbations are performed based on which gRNA the cell receives. These gRNAs are typically delivered via lentiviral transduction and integrated into the DNA of target cells, allowing the induced perturbation to be efficiently determined based on the gRNA sequence. Creative Biogene's stable Cas9-expressing cell lines are useful for sgRNA library screening.

Figure 2. Experimental design for CRISPR screening. (Bock C, et al., 2022)

FAQ

Q: What is CRISPR-Cas9?

A: CRISPR-Cas9 is a revolutionary gene-editing technology that allows scientists to precisely modify genes within an organism's DNA. CRISPR stands for "Clustered Regularly Interspaced Short Palindromic Repeats," which are short, repeated DNA sequences found in the genomes of bacteria and other microorganisms. Cas9 refers to the CRISPR-associated protein 9, an enzyme that acts as a molecular scissor.

Q: How does the CRISPR-Cas9 system work?

A: The CRISPR-Cas9 system works by guiding the Cas9 enzyme to a specific target DNA sequence using a small RNA molecule called guide RNA (gRNA). The gRNA is designed to complement the sequence of the target gene, allowing the Cas9 enzyme to bind to that specific location. Once in place, Cas9 cleaves the DNA three bases from the end of the target sequence, thus providing a precise replacement, deletion, or insertion of genomic material.

Q: How does Cas9 know where to cut DNA?

A: The Cas9 protein, obtained from Streptococcus pyogenes, works with a "guide" RNA that targets a complementary 20-nucleotide segment of DNA. Once the RNA recognizes a sequence that matches these nucleotides, Cas9 cuts the double-stranded DNA helix.

Q: Why use CRISPR-Cas9 technology to generate cell lines?

A: CRISPR-Cas9 technology has many benefits for gene editing and cell line creation:

(1) It is three to four times more efficient than conventional systems such as ZFN and TALEN4.

(2) Multiple genes can be easily modified by introducing several different guide RNAs.

(3) Target genes can be precisely modified during knockout cell line creation, completely eliminating the protein they encode.

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* For research use only. Not intended for any clinical use.

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