【Star of the Month】Human Epigenetic Knockout Library, Mouse & Human Whole Genome Knockout/ Activation Library

CRISPR library screening is a high-throughput gene screening solution based on CRISPR/Cas9 technology. It constructs a library containing thousands of sgRNAs and clones these sgRNAs into lentiviral vectors to infect target cells at a low MOI. This method can ensure that each cell is infected with only one sgRNA, thereby enabling functional screening of genes corresponding to different sgRNAs. Here, EDITGENE is recommending you the three best-selling CRISPR library spot plasmids this month and attaching an interpretation of the relevant research literature of the library, hoping it will contribute to your path of scientific research.

1. In vivo CRISPR library screening identifies Asf1a as an immunotherapy target for Kras mutant lung adenocarcinoma

Link to original text : https://doi.org/10.1158/2159-8290.CD-19-0780

Lung adenocarcinoma (LUAD) is a highly lethal cancer in which KRAS and EGFR are the major oncogenes. Although LUAD patients with EGFR mutations can be cured by molecularly targeted therapies, KRAS mutations remain a difficult target for direct inhibitors to capture and require the development of new therapeutic strategies. The role of epigenetic regulators in the tumor immune microenvironment is gradually being recognized, and there is increasing evidence that epigenetic factors are involved in regulating the tumor immune microenvironment and may be potential targets for modulating the anti-tumor immune response. CRISPR library screening is a new approach for rapid identification of genes that affect the tumor immune microenvironment.

The researchers constructed an epigenetic gene knockout library containing 524 epigenetic regulators and 173 control genes (including essential genes and immune regulators), which was used in an in vivo CRISPR screen for Kras G12D/Trp53. −/− (KP) LUAD model to identify epigenetic regulators that enhance tumor sensitivity to anti-PD-1 treatment. Screening data results show that the loss of the histone chaperone Asf1a in tumor cells can make tumors more sensitive to anti-PD-1 treatment. The loss of Asf1a promotes the differentiation of M1-like macrophages and enhances T cell activation, augmenting immune responses. Through epigenetic gene knockout library screening , a new therapeutic target Asf1a was discovered, which provides a potential therapeutic target for KRAS-mutant LUAD patients and is of great significance for the development of new cancer treatment strategies.

Figure 1 Screening of the Epigenetic knockout library identifies Asf1a as a negative regulator of anti-pd -1 treatment response

2.The whole mouse genome knockout library facilitates research on the regulatory mechanism of Hedgehog signaling

Link to original text: https://doi.org/10.1101/699819

The HH signaling pathway plays an important role in embryonic development, tissue regeneration, and tumorigenesis. The G protein-coupled receptor family protein Smoothened (SMO) is a key mediator of HH signaling. It transmits signals across the cell membrane through direct interaction with cholesterol. However, the question of how cholesterol is precisely regulated to control signaling systems that may lead to birth defects and cancer has not been addressed.

In this article, researchers used the Brie Mouse CRISPR Knockout Pooled library as a screening tool. The researchers used the NIH/3T3-CG cell line, which expresses Cas9 and contains a fluorescent reporter gene (GLI-GFP) responsive to HH signaling, and the cells were cultured in low-serum and U18666A conditions to ensure sensitivity to perturbations in endogenous lipid metabolic pathways. The screen successfully identified all four positive controls: Smo and Adrbk1 (or Grk2) as positive regulators of the Hh signaling pathway, and Ptch1 and Sufu as negative regulators. A murine genome-wide knockout library revealed the positive regulatory effect of mutations in genes in the cholesterol biosynthesis pathway on the HH signaling pathway, identifying genes in the sphingolipid biosynthesis pathway as HH negative regulator of signaling strength, providing genetic evidence for understanding how cholesterol acts as an endogenous steroidal lipid to regulate SMO activation, which is important for developing potential strategies for treating related diseases.

Figure 2 CRISPR screening identifies lipid-related genes that affects Hedgehog signaling

3. Human genome-wide activation library screening for key lncRNAs involved in the regulation of sorafenib response in HCC

Link to original text : https://doi.org/10.1186/s12943-024-01988-y

Hepatocellular carcinoma (HCC) is the fifth deadliest cancer worldwide. Sorafenib, the first FDA-approved first-line agent for the systemic treatment of advanced and end-stage HCC, is a tyrosine kinase inhibitor that blocks enzyme activity relevant to HCC growth and proliferation and exhibits anti-angiogenic effects, thereby prolonging the overall survival of HCC patients. Common mechanisms of sorafenib resistance include changes in transporter proteins and drug targets, as well as variants in signaling pathways. Growing evidence suggests that non-coding RNAs, including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), are important regulators of biological processes in HCC, and in order to identify key lncRNAs involved in the regulation of sorafenib response in HCC, the researchers carried out a related study.

The researchers used CRISPR/Cas9 synergistic activation medium (SAM) libraries to screen for key lncRNAs regulated by sorafenib treatment. The results showed that lncRNA LINC01056 was one of the most significantly down-regulated lncRNAs in sorafenib-resistant HCC cells and that knockdown of Linc01056 (KD) decreased HCC cellular sorafenib sensitivity, inhibits apoptosis in vitro, and promotes tumor growth in mice in vivo. Mechanistically, the researchers identified PPARα as a key molecule regulating metabolic switching in HCC cells after Linc01056 knockdown, and PPARα inhibition restored the response of HCC cells and tumors to sorafenib. Human CRISPR/Cas9 SAM pooled libraries revealed the role of Linc01056 as a key epigenetic regulator and potential therapeutic target in modulating sorafenib response in HCC, providing possible directions for the development of new therapeutic strategies against sorafenib-resistant HCC.

CRISPR screen identifies Linc01056 as a regulator of sorafenib response in HCC cells

EDITGENE have off-the-shelf libraries used in the above studies, with coverage ≥ 99% and homogeneity <10, with NGS sequencing reports attached. The off-the-shelf libraries can be delivered within one week! There are also plasmids/viruses from the most popular libraries for scientific research~

You may want to check out:

[Literature Review] Improving the Sensitivity of LbuCas13a Detection using Engineered crRNAs

The Knits and Grits behind single-cell CRISPR screening

[MAR Bestselling] CRBN and Myc knockout cell lines; CRISPR whole genome library