Cas9 Stable Cell Lines

1. Stable Knockout Cell Line Generation info@creative-biogene.com Website: https://www.creative-biogene.com

2. Brief Introduction Genomic engineering in cell lines is a versatile tool for studying gene function, designing diseases models, biopharmaceutical research, drug discovery and many other applications. CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems is a newly developed yet the most popular method for genome editing. It has been widely used in current biology, functional genome screening, cell-based human hereditary disease modeling, epigenomic studies and visualization of cellular processes.

3. Brief Introduction CRISPR/Cas9 system consists of a “guide” RNA (gRNA) and a bacterial CRISPR-associated endonuclease (Cas9). The gRNA is a short synthetic RNA composed of a Cas9-binding “scaffold” sequence and ∼20 nucleotide “targeting” sequence that defines the target genomic site to be modified. Cas9 contains two nuclease domains to induce site-specific DNA cleavage. It’s a scalable genome-wide editing technology for its ease of generating gRNAs. The simplicity and high-efficiency of CRISPR/Cas9 system make it a preferable genomic knockout method to the traditional ZFN and TALEN system. Our scientists are experts at performing gene knockout with CRISPR/Cas9, from designing gRNA constructs to transfection and single clone generation of a wide range of cells, including difficult-to-transfect and tumor cell lines.

4. Single Gene Knock Out Cell Line Generation 1 CONTENTS Multiplexed Gene Knock Out Cell Line Generation 2 Fragment Deleted Stable Cell Line Generation 3

5. Single Gene Knock Out Cell Line Generation CRISPR/Cas9 is a revolutionary technology in the field of gene engineering. It has expedited the course of biopharmaceutical research and drug discovery, by providing scientists with a versatile tool to knockout any gene, in any cell and without introducing foreign DNA. The benefits of CRISPR/Cas9 over previous forms of gene editing, such as TALENs and zinc finger nuclease (ZFN), are that it is much simpler to implement and has higher efficiency at performing bi-allelic gene modifications. Creative Biogene is experienced to provide a full CRISPR/Cas9-based single gene knockout service using any mammalian cell line and targeting any gene. Our scientists are experts at performing gene knockout with CRISPR/Cas9, from designing gRNA constructs to transfection and single clone generation of a wide range of cells, including difficult-to-transfect and tumor cell lines.

6. Single Gene Knock Out Cell Line Generation Our services include:

7. Multiplexed Gene Knock Out Cell Line Generation • Gene families or biological pathways usually contain several related genes that collaborate in the same role. Increasing demand in loss-of-function screens has warranted the need for a multiplexed gene knock out cell line. Multiplexing with CRISPR/Cas9 can accelerate genome engineering efforts in biopharmaceutical research and drug discovery. • Creative Biogene provides additional rounds of transformation and selection until every targeted gene is knocked out. Mammalian cells exposed to two or three gRNAs in turn resulted in the generation of cell lines harboring double or triple gene knockouts, that is, the successful disruption of two or three genes. All multiplexed knockout events were obtained with DNA mutations in targeted genes, and harbored the expected knockout phenotype(s). Investigation of entire gene families or biological pathways is enabled by our dual/triple gene knock out cell line generation service.

8. Multiplexed Gene Knock Out Cell Line Generation • The KO status of each cell line is thoroughly tested as follows • Biallelic mutation/deletion confirmed by PCR and DNA sequencing. • KO stability guaranteed up to 20 passages. • Both products enable unbiased, powerful loss-of-function screens. Applications • Targeted screening of specific pathways of importance. • Thorough screening of pathway or disease-focused genes. • Used as screening tools in the development of drugs. • To understand the mechanism of action of a drug. • To target specific biological processes.

9. Multiplexed Gene Knock Out Cell Line Generation Service Case

10. Fragment Deleted Stable Cell Line Generation CRISPR/Cas9 has been widely utilized to facilitate functional knockout of protein coding genes. However, a single site of gene editing may not be sufficient to disrupt protein’s structure (for example, miRNA stem-loop) and function. To overcome this potential limitation, paired guide RNAs targeting both 5’ and 3’ of the target region have been shown to result in larger deletions, thus resulting in loss-of-function completely. • Dual sgRNA-directed large gene deletion mediated by CRISPR/Cas9 system is a robust tool for generating deletions of long non-coding RNAs (lncRNAs), MicroRNAs (miRNAs) and regulatory sequences. Several recent studies have reported the CRISPR/ Cas9 system-induced large genomic deletion, ranged from 23kb to 1Mb, in mouse, C. elegans, rabbit and human cell lines.

11. Fragment Deleted Stable Cell Line Generation Creative Biogene is experienced in producing a fragment deleted cell line using any mammalian cell line and targeting sequences of your interest. Utilization of two sgRNAs targeting the immediate sequence upstream and downstream is a viable method for functional knockout of a target protein completely.

12. Advantages • Our CRISPR gene knockout technology • applies to targets of any genes and • mammalian cells. • One-stop service is provided from design of gRNAs, cell transfection, preparing of single clones to sequencing analysis, only needing customers to provide target genes information and the cell line of interest. • Professional researchers • have rich cell culture experience and • plentiful CRISPR genome editing experience. • Cell lines can be made from nearly • any cell type, including cells that are • difficult to transfect and grow incl. primary cells.

13. THANKS！ info@creative-biogene.com Website: https://www.creative-biogene.com