[Weekly News] CRISPR Library Screening: The Key Role of EV-DNA Structure in Kupffer Cell Mediated Antitumor Immunity

CRISPR/Cas technology is a revolutionary tool in modern biological sciences, with applications spanning medicine, agriculture, environmental conservation, and more. New findings and case studies continue to emerge across these fields. Our ‘CRISPR Weekly News’ column brings you the latest research and industry updates. Here's a brief summary of the past week's highlights:

I. Research Updates

i. CRISPR Screening

1. Title：Unique structural configuration of EV-DNA primes Kupffer cell-mediated antitumor immunity to prevent metastatic progression

Journal：Nature Cancer (IF: 23.5)

Original Link：https://doi.org/10.1038/s43018-024-00862-6

Tumor metastasis is the leading cause of cancer-related death, and the host immune system plays a crucial role in antitumor metastasis. Extracellular vesicles (EVs) are small membrane vesicles released by cells that can carry biomolecules such as proteins, RNA, and DNA for transmission of intercellular information. Kupffer cells are resident macrophages in the liver and are key cells for immune defense, but their mechanisms in resisting metastasis are not fully understood.

Researchers have demonstrated that EV-DNA is primarily located on the surface of vesicles and is associated with uniquely modified and cleaved histones. In order to identify the functional EV-DNA fragments, the related receptors or pathways, and optimize experimental models, researchers conducted the CRISPR whole genome knockout screening , the results showed that immune developmental pathways and genes, including apoptotic peptidase activating factor 1 (APAF1) and neutrophil cytoplasmic factor 1 (NCF1), regulate the packaging of EV-DNA .

Additionally, in the colorectal cancer model, the absorption of EV-DNA by liver Kupffer cells (KC) prior to metastasis activated DNA damage response. This activation reconnected the production of KC cytokines and promoted the formation of tertiary lymphoid structures for inhibiting liver metastasis. On the contrary, the absence of APAF1 reduces the packaging of EV-DNA and promotes liver metastasis. The secretion of EV-DNA in colorectal cancer biopsy can serve as a predictive biomarker for postoperative metastasis, and the unique chromatin of EV-DNA can induce antitumor immunity.

2. Title：UBA1 inhibition sensitizes cancer cells to PARP inhibitors

Journal：Cell Reports Medicine (IF：11.7)

Original Link：https://doi.org/10.1016/j.xcrm.2024.101834

PARP inhibitors are a class of targeted therapeutic drugs targeting DNA damage response (DDR) related deficient tumors, such as BRCA1/2 mutant tumors. However, many cancer cells are resistant to PARP inhibitors due to the compensatory mechanisms of DNA repairing function or other survival pathways. UBA1 is the initiating enzyme of ubiquitination pathway, responsible for activating ubiquitin molecules.

In order to explore whether UBA1 inhibition can weaken the DNA repair ability of cancer cells and enhance their sensitivity to PARP inhibitors, researchers used whole genome CRISPR knockout screening and found that the consumption of ubiquitin activating enzyme E1 (UBA1) enhanced the sensitivity of ovarian cancer cells with HR normal function to PARPi. Studies have shown that silencing or pharmacological inhibition of UBA1 can make multiple cell lines and organ models sensitive to PARPi. Mechanism studies have found that UBA1 inhibition not only hinders HR repair to make cells sensitive to PARP inhibition, but also increases PARylation, and subsequently PARP inhibition may target the PARp modification.

The study revealed the new mechanism of ubiquitination system in DNA repair and tumor drup resistance, and emphasized that UBA1 is a potential target for regulating DNA repair and enhancing antitumor efficacy. This strategy is expected to achieve clinical transformation in ovarian cancer, breast cancer, prostate cancer and other cancers.

ii. CRISPR Knockout Cell Lines

1.Title：Identification of SLC35A1 as an essential host factor for the transduction of multi-serotype recombinant adeno-associated virus (AAV) vectors

Journal：ChemBioEng Reviews (IF:6.2)

Original Link：https://doi.org/10.1128/mbio.03268-24

AAV vectors are the most commonly used delivery tool in gene therapy due to their safety and efficacy, targeting various tissues and cell types, but their efficiency is influenced by serotypes and host cytokines. In order to identify the key host factors that are common in various serotypes of AAV infection and elucidate their mechanisms of action, CRISPR-Cas9 gene knockout libraries were used to perform whole genome screening of host cells, searching for the crucial genes for AAV infection. After two rounds of screening, the highly screened genes were discovered, including KIAA0319L, TM9SF2, and RNF121 previously reported, as well as a group of genes involved in polysaccharide biosynthesis, Golgi apparatus localization, and endoplasmic reticulum penetration.

This study is contructed by CRISPR gene knockout, Overexpression and RNA interference were exploring the function of solute carrier family 35 member A1 (SLC35A1). The results showed that knockout of SLC35A1 significantly reduced the transduction efficiency of AAV, revealing SLC35A1 as a key factor in AAV multi-serotype transduction and expanding our understanding of AAV infection mechanisms. By regulating SLC35A1 or sialic acid metabolism, it may enhance the transduction efficiency of AAV in target cells.

2.Title：Glycosylation of Ceramide Synthase 6 is Required for Its Activity

Journal：Journal of Lipid Research (IF:5)

Original Link：https://doi.org/10.1016/j.jlr.2024.100715

Ceramide Synthase (CerS) is a key enzyme that catalyzes the generation of ceramides, and its functions involve lipid metabolism, cell signal transduction, and apoptosis regulation. And CerS6 mainly generates the ceramides containing C16 fatty acid chains, which are closely related to tumor progression, metabolic disorders, and neurodegenerative diseases.

In order to investigate whether CerS6 is glycosylated and its effects on enzyme activity, structural stability, and cellular function, researchers used CRISPR/Cas9 technology to knock out the CerS6 gene and perform enzyme activity detection, glycosylation effect verification, and cellular functional research on cells.

The results showed that after knocking out CerS6, the enzyme activity was completely lost. Re-expression of wild-type CerS6 could restore enzyme activity, while glycosylation mutants (such as N/Q mutants) could not, indicating that glycosylation is crucial for CerS6's activity in catalyzing ceramide synthesis. Glycosylation may maintain its activity by affecting protein folding, enzymatic substrate binding, or stability in the endoplasmic reticulum.

The study reveals new functions of glycosylation and provides a new mechanism for how glycosylation affects the generation of ceramides, providing a new perspective for understanding the key regulatory links in lipid metabolism disorders.

iii. CRISPR Detection

1.Title：An Electrochemical Biosensor for the Detection of Tuberculosis Specific DNA with CRISPR-Cas12a and Redox probe Modified Oligonucleotide

Journal：Heliyon (IF: 3.4)

Original Link：https://doi.org/10.1016/j.heliyon.2024.e40754

The development of a powerful and accurate real-time detection platform for tuberculosis (TB) biomarkers is crucial for disease control. In current study, the detection principle depends on the crushing of PES modified non-specific ssDNA (Poly T) in the presence of target DNA IS6110, which is a reliable biomarker for tuberculosis diagnosis through CRISPR-Cas12a mechanism. Cas protein has great potential in nucleic acid detection.

Researchers utilize CRISPR/Cas12a to develop a biosensing platform based on its trans-cleavage activity and applied to electrochemical biosensors. In the presence of target DNA, trans cleavage activity was observed through non-specific substrate PolyT chains of ssDNA. Different concentrations of target DNA were tested on the constructed biosensor, and the fabricated biosensor successfully detected TB target DNA through trans-cleavage of polyT modified with PES.

This new biosensor can detect the target DNA IS6110 within 60 minutes through the trans-cleavage activity of CRISPR/Cas12a, with a detection limit of 14.5 nM. The results reveal the potential of Cas12a based biosensors as diagnostic platforms. The electrochemical biosensor based on CRISPR/Cas12a endonuclease developed by researchers provides a potential and powerful platform for accurate detection of Mycobacterium tuberculosis.

2.Title：End-point RPA-CRISPR/Cas12a-based detection of Enterocytozoon bieneusi nucleic acid: rapid, sensitive and specific

Journal：BMC Veterinary Research (IF: 2.3)

Original Link：https://doi.org/10.1186/s12917-024-04391-3

Intestinal cell parasites are a common type of microsporidia that infect humans and animals. Chinese researchers have validated a CRISPR/Cas12a based diagnostic detection method for intestinal parasites (ReCTC). The detection limit of this method reaches 3.7 copies/µ l, showing high sensitivity and specificity to other intestinal pathogens. ReCTC outperforms nested PCR, which can effectively identify intestinal parasites in clinical samples, demonstrating the potential for rapid and on-site application, providing a promising tool for clinical diagnosis.

3.Title：HRP-Integrated CRISPR/Cas12a Biosensor for Rapid Point-of-Care Detection of Langya Henipavirus

Journal：iScience (IF:4.6)

Original Link：https://doi.org/j.isci.2024.111466

The Langya henipavirus (LayV) biosensor based on CRISPR-Cas12a can achieve ultra sensitive RNA detection, which is capable of detecting 10 samples/μ L by RPA and also capable of detecting 1200 samples/μ L by horseradish peroxidase (HRP) - ssDNA reporter gene without amplification. This fast, accurate, and amplicon-free method provides a promising real-time diagnostic approach for emerging pathogens, especially in environments with limited resource.

Langya henipavirus (LayV) is an emerging zoonotic pathogen that has the potential to cause a pandemic, but we are currently at the lack of rapid diagnostic tools, especially for immediate diagnosis. Therefore, researchers have utilized the advantages of CRISPR/Cas12a biosensing to establish a highly sensitive LayV detection method. By combining CRISPR/Cas12a with RPA, ultra sensitive detection of LayV RNA at 10 copies/μ L was achieved within 30 minutes at room temperature.

In addition, researchers have developed a special horseradish peroxidase (HRP) - ssDNA reporter gene that enables CRISPR/Cas12a to detect LayV RNA without pre-amplification, achieving a sensitivity of 1200 copies/μ L, which is detectable by the naked eyes. These explorations can serve as accelerators for CRISPR/Cas biosensing to quickly respond to emerging biological threats and provide a method for achieving simple, accurate, and amplicon-free for real-time pathogen screening in resource limited or underdeveloped areas.

iv. Other CRISPR-Related Research

1. Title：Genome editing with the HDR enhancing DNA PKcs inhibitor AZD7648 causes large-scale genomic alterations

Journal：Nature Biotechnology (IF: 33.1)

Original Link：https://doi.org/10.1038/s41587-024-02488-6

DNA-PKcs (DNA dependent protein kinase catalytic subunit) is an important protein closely related to the NHEJ repair pathway, which participates in the repair process after DNA double strand breaks. If DNA-PKcs is inhibited, the utilization of HDR pathway can be increased, thereby the accuracy of gene editing is improved.

AZD7648 is an inhibitor of DNA-PKcs known to promote the HDR pathway by inhibiting NHEJ to improve the efficiency and accuracy of gene editing. In order to investigate the impact of AZD7648 on genome editing, especially whether it causes large-scale genomic changes by enhancing the HDR pathway, researchers used AZD7648 to inhibit DNA-PKcs during CRISPR gene editing in human cell lines (such as HEK293 or K562 cells) and mouse models, for enhancing the HDR effect and evaluating the changes of edited genome through the methods of genome sequencing, fluorescence microscopy observation, these changes include insertions, deletions, point mutations, etc.,. The researchers further investigated whether AZD7648 induces unexpected large-scale genomic changes while enhancing HDR.

The results showed that using DNA-PKcs inhibitor, AZD7648, for CRISPR/Cas9 editing can enhance the homology directed repair, but induces kilobase and megabase level deletions, translocations, and chromosome arm loss. These genomic changes can evade the detection through standard sequencing, so caution is needed in clinical applications. Combining AZD7648 with PolQi2 can reduce a large number of deletions while maintaining HDR efficiency, but the challenge of megabase level events still exists.

2.Title：Engineered transcription-associated Cas9 targeting in eukaryotic cells

Journal：Nature Communications (IF：14.7)

Original Link：https://doi.org/10.1038/s41467-024-54629-9

The traditional CRISPR/Cas9 system uses DNA cleavage for gene editing, but this method may lead to unstable genomic changes and non-specific mutations, therefore there is an urgent need to develop the new technologies to avoid these problems. To overcome this challenge, researchers fused Cas9 protein with transcription activating factors (such as VP64 and p300) to develop a transcription related Cas9 system that directly regulates gene transcription by binding to target DNA sequences without DNA cleavage.

In terms of methodology, researchers employed the binding of dCas9 (endonuclease deficient Cas9) to transcription factors to achieve the transcriptional activation of target genes. Based on the experiments in multiple eukaryotic cell lines, the efficiency and specificity of this system have been validated, demonstrating its ability to significantly increase the expression levels of specific genes without causing DNA damage.

The research results indicate that this non-cleaving gene activation system can effectively activate the transcription of target genes, avoiding the genomic instability problems caused by traditional gene editing techniques, with high targeting ability. This technology not only provides new ideas for gene therapy, especially for treatment strategies that require activating gene expression rather than knocking out genes, but can also be applied to the construction of disease models and the development of synthetic biology.

II. Industry News

1. Intellia Therapeutics announced that the U.S. Food and Drug Administration (FDA) has granted Regenerative Medicine Advanced Therapy (RMAT) designation to its gene editing therapy NTLA-2001 for the treatment of hereditary ATTR amyloidosis. NTLA-2001 is a CRISPR/Cas9 based therapeutic approach aimed at reducing the production of abnormal proteins that cause diseases by targeting the TTR gene. Obtaining RMAT designation means that its therapy can accelerate the clinical development with the priority and support from the FDA in clinical trials, approvals, and other aspects. This designation further advances Intellia's leading position in the field of gene therapy and provides new hope of treatment for genetic diseases.

News Link： https://ir.intelliatx.com/news-releases/news-release-details/intellia-therapeutics-announces-fda-regenerative-medicine-0

2. Scribe Therapeutics recently addressed its preclinical data at American Heart Association (AHA) Scientific Sessions 2024, validating the potential of its CRISPR genome editing and epigenome modifying technology in the treatment of cardiometabolic diseases. The ELXR platform achieved a 67% reduction in LDL-C within 6 months in non-human primates through epigenetic silencing. The XE platform reduced APOC3, triglycerides, and cholesterol by over 90%, 97%, and 84%, respectively, with high specificity and no off-target effect. These preclinical data provide the strong support for Scribe to apply its gene editing technology to treat metabolic diseases in the future. The application of this platform not only promotes gene therapy for metabolic diseases, but also lays the foundation for further clinical trials, demonstrating its innovative potential for cardiovascular diseases.

News Link： https://www.businesswire.com/news/home/20241122617151/en/Scribe-Therapeutics-Reports-Preclinical-Data-Validating-its-CRISPR-Genome-Editing-and-Epigenome-Modifying-Technologies-for-Addressing-Cardiometabolic-Disease-at-American-Heart-Association-AHA-Scientific-Sessions-2024

EDITGENE focuses on CRISPR technology, offering a range of high-quality gene editing services and in vitro diagnostic products. These include but are not limited to：CRISPR Library Screening、Cell Line Engineering、Monoclonal Cell Line Screening、CRISPR Detection.We are committed to providing the most efficient technical services for CRISPR-related, gene function research, in vitro diagnostics, and therapeutic research.