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：Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes

Journal：Nature Biomedical Engineering  (IF: 26.8)

Original Link：https://doi.org/10.1038/s41551-024-01278-4

Most Whole Genome CRISPR Screening usually use hybrid libraries based on single guide RNA (sgRNA) vectors. However, some important phenomena in cell biology, such as intercellular interactions or secretion of bioactive molecules, cannot be studied through CRISPR hybrid libraries only. To overcome this limitation, researchers successfully constructed an arrayed library using large-scale parallel plasmid cloning methods for genome-wide knockout of human protein-coding genes (including 19,936 plasmids) and activation and epigenetic-silencing of genes (including 22,442 plasmids).

Each plasmid encodes four non-overlapping sgRNAs, designed to tolerate the most polymorphisms in human DNA. Large-scale parallel cloning technology enables the creation of four-sgRNA CRISPR libraries for whole-genome gene knockout, activation, and silencing, covering approximately 20,000 human genes. These libraries can achieve efficient gene perturbation, with knockout efficiencies ranging from 75% to 99% and silencing efficiencies from 76% to 92%.

Researchers utilized a CRISPR arrayed library containing four sgRNAs to significantly enhance the efficiency of genetic perturbations. This approach demonstrated high efficacy in gene knockout and silencing studies, while also producing substantial fold changes in gene activation experiments, showcasing its potential for comprehensive genome editing applications. In addition, 11 new PrPC regulatory factors were identified in the array activation screening of 1,634 human transcription factors, and 5 new autophagy-modifying factors were identified through the aggregation screening of the ablation library, which were not found in traditional screening methods. In summary, the arrayed library of four sgRNAs provides a powerful and multifunctional resource for whole-genome perturbation research, revealing new cellular biology mechanisms and promoting research progress in related fields.

2. Title：Straight to the heart: Protecting the patient's heart during chemotherapy

Journal：Cell Stem Cell  (IF: 19.8)

Original Link：https://doi.org/10.1016/j.stem.2024.10.010

Chemotherapy is an important means of treating cancer; however, many chemotherapy drugs, especially certain types such as Doxorubicin and Cyclophosphamide, are toxic to the heart and may cause chemotherapy-related cardiac toxicity (CRT). These drugs can damage the myocardium, leading to cardiovascular problems such as heart failure and arrhythmia, and in severe cases, may even endanger life. Therefore, protecting patients' hearts from the harmful effects of chemotherapy has become an important clinical challenge.

In order to understand the toxic effects of chemotherapy drugs on the heart and identify genes or molecules that can protect the heart, researchers used a whole-genome CRISPR library to discover multiple potential cardioprotective factors. These factors may exert their effects by regulating cellular stress responses, repair mechanisms, or by reducing heart damage. For example, genes related to oxidative stress, mitochondrial function, the inflammatory response, or autophagy may have been identified, which are potential mechanisms of chemotherapy-induced cardiac toxicity. 

Additionally, new regulatory factors related to PrPC (cellular prion protein) and autophagy have been discovered. PrPC is associated with neurodegenerative diseases and may also affect the survival and function of heart cells; autophagy is a protective cellular mechanism that can clear damaged organelles, proteins, or debris. The research findings suggest that these factors may provide new targets for cardiac protection.

This study utilizes CRISPR library screening technology to harness the power of large-scale gene perturbations in discovering cardioprotective mechanisms associated with chemotherapy. Through multiple screening methods, such as gene knockout, activation, and silencing, researchers have identified new genes and pathways that could serve as targets for developing cardioprotective drugs. By optimizing screening techniques (e.g., ablation libraries, post-aggregation of lentiviruses), the accuracy and reproducibility of results are ensured, advancing research in this field.

ii. CRISPR Knockout Cell Lines

1.Title：Integrative analysis identifies the atypical repressor E2F8 as a targetable transcriptional activator driving lethal prostate cancer

Journal：Oncogene（IF: 6.9）

Original Link：https://doi.org/10.1038/s41388-024-03239-2

Members of the E2F family typically play critical roles in cell cycle regulation, gene expression, and tumorigenesis. Although most E2F family members are considered transcriptional activators, E2F8 is referred to as an 'atypical' suppressor. Early studies showed that E2F8 primarily regulates cell proliferation by inhibiting the expression of cell cycle genes. However, recent studies have demonstrated that E2F8 not only has inhibitory effects but also acts as a transcriptional activator in certain types of cancer.

The researchers employed an integrated analysis strategy, combining transcriptomics, epigenomics, and clinical data to investigate the role of E2F8 in prostate cancer. Specifically, they utilized CRISPR/Cas9 knockout technology (https://editxor.com/index/service/info/id/17.html) to knock out the E2F8 gene in prostate cancer cells, further confirming its role in cancer progression. The results showed that knocking out E2F8 led to growth arrest and reduced metastatic ability of prostate cancer cells. In clinical samples, high expression of E2F8 was associated with poor prognosis. 

The study validated the key role of E2F8 in prostate cancer through CRISPR/Cas9 knockout technology, revealing that it is not only a transcriptional repressor but also a transcriptional activator that promotes cancer cell proliferation, migration, and invasion. Targeting E2F8 with therapeutic strategies may provide new directions for the treatment of prostate cancer, especially in advanced and metastatic stages.

2.Title：HSP60 inhibits DF-1 apoptosis through its mitochondrial signal peptide

Journal：Poultry Science（IF: 3.8）

Original Link：https://doi.org/10.1016/j.psj.2024.104571

HSP60 participates in many biological functions and plays a critical role in maintaining oxidative stress and mitochondrial integrity. Previous studies have shown that HSP60 has the ability to inhibit cell apoptosis. Based on this, the current study further explores the mechanism by which HSP60 inhibits apoptosis. First, an HSP60 knockout DF-1 cell line (DF-1-HSP60-KO) was established using the CRISPR/Cas9 system, and the apoptosis level of the DF-1-HSP60-KO cell line was measured using flow cytometry and an ELISA apoptosis assay kit. Next, the effect of HSP60 knockout on relevant apoptotic factors was analyzed by Western blotting and RT-PCR. The results showed that, compared with control DF-1 cells, HSP60 knockout cells had a significantly increased apoptosis rate, decreased Bax expression, and increased Caspase-3 expression. HSP60 knockout induces cell apoptosis by upregulating Caspase-3 and downregulating Bax expression. 

Using PyMOL software to predict the structure of the HSP60 mitochondrial signal peptide (MIT) protein, it was found that the 21st His amino acid affects its spatial structure. Additionally, the HSP60 mutant protein (TB) was transfected into DF-1-HSP60-KO cells and induced by Bardoxolone Methyl. Compared to the wild-type HSP60 group, the apoptosis rate was significantly increased, the cell survival rate was decreased, and differential changes in genes such as Bax, Bak, and Bcl-2 were observed.

In summary, the 21st His amino acid in the HSP60 MIT inhibits cell apoptosis by regulating the levels of genes involved in the apoptosis signaling pathway. This study reveals the role of HSP60 in regulating cell apoptosis through mitochondrial signaling peptides, which has potential therapeutic value.

iii. CRISPR Detection

1.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

Enterocytozoon bieneusi is a common microsporidian that can infect humans and other animals. Current methods for detecting Enterocytozoon bieneusi infections suffer from deficiencies in sensitivity, specificity, simplicity, cost, and speed, making them unsuitable for clinical application.Researchers have extended the ReCTC method and established an E. bieneusi nucleic acid detection technique targeting the swp gene. 

They first evaluated the ReCTC method, based on CRISPR/Cas12a, which was previously reported, and tested its effectiveness in detecting E. bieneusi nucleic acid. Sensitivity (LOD) and specificity were then assessed using prepared target DNA. Finally, the study validated the accuracy of the ReCTC-based method in detecting E. bieneusi in clinical samples.

The ReCTC method has been successfully applied for nucleic acid detection of E. bieneusi. Sensitivity testing showed that the LOD of the fluorescence and lateral flow test strip methods (based on ReCTC) was 3.7 copies/µl. Specificity tests involving other common intestinal pathogens revealed that fluorescence signals and/or test lines appeared only when the sample was positive for E. bieneusi. These results indicate that the ReCTC method can successfully detect E. bieneusi in clinical samples. With high sensitivity and strong specificity, the ReCTC method outperforms nested-PCR in clinical DNA samples. Additionally, it has demonstrated the ability for on-site detection.

2.Title：Single-tube detection of a foodborne bacterial pathogen using user-friendly portable device

Journal：Biosens Bioelectron（IF: 10.7）

Original Link：https://doi.org/10.1016/j.bios.2024.117035

Foodborne diseases are a major global public health issue, causing a significant number of illnesses and deaths. To effectively control the spread of foodborne pathogens, researchers are working on developing a portable device capable of rapid detection of foodborne bacterial pathogens in a single-tube reaction. This device must not only have high sensitivity and specificity, but also ensure ease of use, enabling non-professionals to operate it. It should be suitable for practical applications, such as food safety testing.

After systematically studying the compatibility of protein nucleic acid signal transduction, exponential amplification reaction (EXPAR), CRISPR, and the advantages of CRISPR/Cas12a, researchers have developed a "three-in-one" integrated reaction called ST-EXPAR-CCRISPR detection. This portable device, based on CRISPR technology, can quickly and accurately detect foodborne bacterial pathogens in a single-tube reaction. It offers a convenient and efficient detection tool for food safety, with broad application prospects, particularly for on-site rapid detection and public health emergency response. Additionally, the ST-EXPAR-CCRISPR detection method can be easily modified by changing the nucleic acid sequence to detect other targets, with low research and development costs, and the potential to reduce the global disease burden.

iv. Other CRISPR-Related Research

1. Title：Enhanced osteogenic potential of iPSC-derived mesenchymal progenitor cells following genome editing of GWAS variants in the RUNX1 gene

Journal：Bone Research (IF: 14.3)

Original Link：https://doi.org/10.1038/s41413-024-00369-x

Bone development and regeneration is a complex biological process involving multiple genes and cell types. During bone formation, mesenchymal precursor cells (MPCs) play a crucial role, as they can differentiate into osteoblasts and promote bone formation. RUNX1 (Runt-related transcription factor 1) is a key transcription factor known to be involved in various biological processes, including blood and bone development. Recent genome-wide association studies (GWAS) have identified 518 important loci associated with bone mineral density (BMD), including variants in the RUNX1 locus (rs13046645, rs2834676, and rs2834694). 

However, the regulatory effects of these variants on RUNX1 expression and bone formation remain unclear. Researchers used CRISPR/Cas9 genome editing technology to repair or knock in the GWAS variants in the RUNX1 gene related to bone formation in iPSC-derived MPCs. They evaluated whether this editing could enhance the bone-forming potential of these cells through in vitro bone formation experiments (such as mineralization assays and osteogenic marker detection).

This enhanced bone regeneration ability offers new insights for treating bone diseases such as osteoporosis and fractures. Additionally, the study revealed the role of RUNX1 gene mutations in bone formation, further deepening our understanding of bone development and disease mechanisms. Researchers aim to improve bone regeneration therapies using this technology, providing new approaches for the treatment of diseases such as osteoporosis and fractures.

II. Industry News

HuidaGene announced the launch of the HG204 clinical trial, with the first patient receiving treatment. On December 6, 2024, HuidaGene Therapeutics announced that the first patient has been dosed in the HERO clinical trial, marking the initiation of the clinical evaluation of the world's first RNA editing therapy (HG204) for the treatment of MECP2 duplication syndrome (MDS). MDS is a rare and fatal neurodevelopmental disorder, and currently, there are no approved disease-modifying therapies for MDS.

HG204 is an RNA editing therapy based on CRISPR/Cas13, which delivers the hfCas13Y editing enzyme and guide RNA targeting the MECP2 gene through a single adeno-associated virus vector, aiming to reduce the overexpression of the MECP2 protein. Preclinical studies have shown that HG204 achieves stable and durable expression in the brain tissue of mice and monkeys, leading to significant improvements in motor skills, social behavior, and survival time. 

The HERO trial is the first clinical trial targeting MDS patients, designed to evaluate the safety, tolerability, and preliminary efficacy of HG204, with a focus on improvements in motor, cognitive, and adaptive behaviors, as well as reductions in MECP2 mRNA and protein levels. MDS primarily affects males and presents with symptoms including intellectual disability, epilepsy, and loss of motor and speech skills. Currently, there is no effective treatment for MDS, but HG204 offers new hope for treating this rare disease

News Link：https://www.huidagene.com/new/news/71-0

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.

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