Knockout cell refers to the use of CRISPR technology for gene editing, through the design of specific sgRNA targeting the cut site of the target gene, so that the Cas9 protein binds to the sgRNA and "cut" the target gene from the cell, thus realizing the gene knockout. Knockout cells have a wide range of applications in life sciences, medicine and drug discovery and development, such as the use of knockout technology to establish disease models, drug screening and target validation, knockout of disease-causing genes to achieve gene therapy for diseases, and so on.

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Advantages of Knockout Cell Lines in Therapeutic Development

Knockout cell lines play a crucial role in target validation, confirming the functional relevance of specific genes in disease pathways. By eliminating the expression of a target gene, researchers can assess its impact on cellular phenotypes, signaling pathways, and disease-related processes, providing evidence to support or refute its therapeutic potential.

• Target validation

Target Identification and Validation: Knockout cell lines play a crucial role in identifying and validating potential therapeutic targets. By disrupting specific genes implicated in disease pathways, researchers can assess the effects of gene loss on cellular phenotypes and disease-related processes. This information helps confirm the functional relevance of targets and guides the development of targeted therapies.

• Understanding disease mechanisms

Knockout cell lines provide insights into the underlying mechanisms of diseases by mimicking the loss-of-function conditions observed in patients. By studying the consequences of gene knockout on cellular processes, researchers can unravel disease pathways, identify key molecular events, and uncover new targets or pathways for therapeutic intervention.

• Drug discovery and development

Knockout cell lines enable the screening and evaluation of potential drug candidates. By incorporating knockout cell lines into high-throughput screening assays, researchers can assess the effects of compounds on disease-related cellular phenotypes, target engagement, and therapeutic efficacy. These cell lines facilitate the identification of lead compounds and the optimization of drug candidates.

• Personalized medicine

Knockout cell lines can be derived from patient samples, allowing for personalized medicine approaches. By creating knockout models that mimic specific genetic alterations observed in patients, researchers can develop tailored therapies and evaluate their effectiveness in patient-derived cell lines. This approach holds promise for precision medicine and individualized treatment strategies.

• Therapeutic resistance and mechanism of action studies

Knockout cell lines aid in the investigation of therapeutic resistance mechanisms and drug mechanisms of action. By studying the effects of target gene knockout on drug sensitivity or resistance, researchers can identify genetic factors influencing treatment response. Additionally, comparing cellular responses in knockout and wild-type cell lines helps elucidate the specific molecular targets and pathways affected by drugs.

Kras Knockout Cells Offer New Avenues for Pancreatic Cancer Treatment

Pancreatic cancer is a highly aggressive and lethal malignancy in which genetic variants of the Kras proto-oncogene are present in 90% of cases, leading to uncontrolled proliferation of cancer cells. Although the battle against pancreatic cancer continues, targeted therapy against Kras has been quite challenging. In recent years, scientists have discovered a gene editing system called CRISPR/Cas9 that offers new hope for cancer treatment.

Researchers knocked down cells carrying the c.35G>A (p.G12D) Kras mutation using the CRISPR/Cas9 system to study and understand the role of Kras mutations in pancreatic ductal adenocarcinogenesis and progression, and verified the knockdown of KrasG12D by DNA sequencing and Western blotting. The results showed that under normal growth conditions, Kras knockout cells were similar to wild-type cells in terms of function and expression. Further study of the key signaling pathways activated by Kras revealed that the hyperactivation of PI3K/Akt in human-derived Kras knockout cells and murine-derived Kras knockout cells showed different expression patterns, which revealed that pancreatic cancer cells have a complexity and diversity of signaling and survival mechanisms. Kras knockout cells will help researchers understand the role of Kras in pancreatic cancer and develop new therapeutic approaches to fight pancreatic cancer more effectively.

Fig. 1 Human and murine Kras knockout cells have different protein expression patterns.

NLRP3 Knockout Cells Reveal Critical Role of Viral Glycoproteins in Triggering Immune Responses

Viral infections and their associated complications have become a major global health problem. Viral infections cause a wide range of diseases with varying symptoms, from asymptomatic to chronic to acute and fatal courses, reflecting the diversity and biology of viruses. In-depth study of viral factors that trigger immune responses is critical to understanding viral pathogenesis. As part of the innate immune system, inflammatory vesicles can be activated by viral pathogens. However, the viral structural components responsible for activating the inflammasome remain largely unknown.

In this study, viruses such as SARS-CoV-1/2, HCMV and HCV were analyzed, and the researchers used CRISPR-Cas9 technology to knock down NLRP3 and GSDMD genes in THP-1 macrophages to understand the roles of these two genes in the inflammatory responses induced by viral glycoproteins. The experimental results showed that viral glycoproteins were able to strongly induce the activation of NLRP3 inflammatory vesicles and cellular pyroptosis, whereas the knockdown of NLRP3 and GSDMD significantly inhibited these responses. These results not only reveal the mechanism of viral glycoproteins as triggers of innate immunity, but also provide new perspectives for vaccine development and vaccine-induced innate immunity studies. NLRP3 knockout cells provide new insights and motivation for understanding viral pathogenesis and developing novel vaccines.

Fig. 2 NLRP3 knockdown inhibits activation of inflammatory vesicles and cellular pyroptosis.

CRISPR-Cpf1-Mediated BRAF Knockout Cell Study Reveals Potential for Precision Cancer Therapy

BRAF-V600E is one of the most reported driver mutations in a wide range of cancers and is present in all patients with hairy cell leukemia, 95% of patients with papillary craniopharyngioma, approximately 45% of patients with papillary thyroid cancer, and more than 50% of patients with melanoma. However, the efficacy of BRAF inhibitors is limited to a subset of patients, and drug resistance and cancer recurrence remain major problems in clinical practice.

The researchers knocked down the V600E-mutant BRAF gene and investigated the gene's role in cancer development and progression by CRISPR-Cpf1 (i.e., CRISPR/Cas12a), CRISPR-Cas9, and CRISPR-Cas9- EQR system technologies, and then compared the intracellular performance and application of the two gene editing systems. The results showed that Cpf1-mediated gene editing was followed by cell death and significant decreases in BRAF and pERK1/2 protein expression, and Cas9 was able to recognize and cleave both normal and mutant alleles, whereas no significant gene editing events were observed in the EQR variant, suggesting that cpf1 has potential applicability in precision medicine. the BRAF knockdown cells provide a useful tool for studying BRAF inhibitor resistance, cancer recurrence, and the development of new therapeutic strategies provides valuable information. By performing gene editing experiments in this cell line, researchers can better understand the promise of Cpf1 in precision medicine, especially in targeting specific cancer-related mutations for gene therapy.

Fig. 3 Cpf1, Cas9 and Cas9-EQR systems and gRNA design of dual fluorescent reporter plasmids for assessing editing efficacy and selectivity.