Unlocking the Potential Exploring Cellular Gene Knockout with CRISPR-Cas9 Technology

The field of bioscience research has been revolutionized by the emergence of CRISPR-Cas9 technology. This groundbreaking tool allows scientists to edit genes with unprecedented precision and efficiency, enabling them to unravel the mysteries of cellular processes and diseases. One of the most powerful applications of this technology is cellular gene knockout. Stable knockout cell lines can be created using advanced CRISPR technology, including single knockout cell lines, multiple knockout cell lines, and fragment deletion stable cell lines. The generated stable cell lines have a wide range of applications, accelerating our understanding of gene function and opening up new possibilities for therapeutic intervention.

Introduction of CRISPR-Cas9 Technology

Known repetitive base sequences called CRISPR ("clustered regularly interspaced short palindromic repeats") were first discovered in 1987 in the genome of Escherichia coli. The CRISPR-Cas9 system consists of two main components: the Cas9 protein and a guide RNA (gRNA). Cas9 is an enzyme derived from bacteria that acts as a pair of molecular scissors, capable of cutting DNA strands at specific locations. gRNA is a short RNA molecule that guides Cas9 to the desired DNA sequence by binding to it, ensuring precise targeting. The mechanism of CRISPR-Cas9 revolves around the ability to edit DNA at specific locations. The gRNA is designed to be complementary to the targeted DNA sequence. When the gRNA binds to its corresponding DNA sequence, the Cas9 enzyme is activated and cuts the DNA double-strand. This cut triggers the cell's natural DNA repair mechanisms, which can lead to either simple mutations or precise modifications introduced by researchers.

Fig. 1 Schematic diagram of the CRISPR-Cas 9 system molecular mechanism. (Janik, E., et al, 2020)

Process for Constructing Knockout Cell Lines Using CRISPR-Cas9

Constructing knockout cell lines using CRISPR-Cas9 technology involves several steps and considerations. Here is an introduction to the general process:

Designing guide RNAs (gRNAs): Determine the target gene to be knocked out and identify suitable gRNA sequences for targeting it. The gRNAs should be specific to the target gene and have minimal off-target effects.
Constructing the CRISPR expression vector: Construct a plasmid or lentivirus-based vector that carries the CRISPR-Cas9 components along with the desired gRNA(s). This vector will serve as the delivery system for introducing CRISPR components into the cell.
Transfection or transduction: Introduce the CRISPR expression vector into the target cells either by transfection or transduction. This can be done using techniques like lipofection, electroporation, or viral transduction.
Selection of CRISPR-edited cells: After introducing the CRISPR components, select for cells that have integrated and expressed the components. This is typically done by including a selectable marker (e.g., antibiotic resistance gene) in the CRISPR expression vector and applying antibiotic selection pressure to eliminate unmodified cells.
Single-cell cloning: Once a population of cells has been selected, single-cell cloning can be performed to establish clonal cell lines. This is important to ensure that each cell line contains a knockout of the target gene, as individual cells within a population may have different edits.
Verification of gene knockout: Confirm the knockout of the target gene in the established clonal cell lines. This can be done using various techniques such as DNA sequencing, Western blotting, or PCR amplification followed by DNA gel electrophoresis.

Application of CRISPR-Cas9 Knockout Cell Line

Unraveling gene function and regulatory pathways.
Investigating disease mechanisms and identifying potential drug targets.
Assessing gene essentiality and functional redundancy.
Uncovering new therapeutic strategies for genetic diseases.

References:

Janik, E.; et al. Various aspects of a gene editing system-crispr-cas9. International Journal of Molecular Sciences. 2020, 21(24): 9604.
Zarei, A.; et al. Creating cell and animal models of human disease by genome editing using CRISPR/Cas9. The journal of gene medicine. 2019, 21(4): e3082.

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

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Unlocking the Potential Exploring Cellular Gene Knockout with CRISPR-Cas9 Technology

Introduction of CRISPR-Cas9 Technology

Process for Constructing Knockout Cell Lines Using CRISPR-Cas9

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