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：Mapping functional elements of the DNA damage response through base editor screens

Journal：Cell Reports（IF: 7.5）

Original Link：https://doi.org/10.1016/j.celrep.2024.115047

DNA damage response (DDR) is a complex signaling network initiated by cells in response to various DNA damages, such as radiation, chemical drugs, replication pressure, etc. This process is crucial for maintaining genomic stability and cell survival. Many cancer-related gene variations are closely related to the disorder of DDR mechanisms, therefore, the study of these mechanisms is an important field in cancer research and treatment.

Researchers constructed a library containing multiple potential DDR gene mutations through whole genome screening of functional lysine residues and genes involved in DNA damage response, and used base editing technology to precisely edit these genes, resulting in different mutations. As a result, it is found that, K494 mutation in C17orf53 disrupts its interaction with RPA protein, leading to increased sensitivity to cisplatin.

In addition, researchers analyzed and identified STK35 as a previously undiscovered gene involved in the DNA damage response (DDR) pathway, indicating that it may play a critical role in DNA repair. This study systematically analyzed the DDR mechanism and revealed the new potential therapeutic targets, providing the valuable clues for cancer research and gene therapy.

2. Title：A multilineage screen identifies actionable synthetic lethal interactions in human cancers

Journal：Nature Genetics（IF: 31.7）

Original Link：https://doi.org/10.1038/s41588-024-01971-9

Common gene mutations in cancer can lead to genomic instability or abnormal signal transduction, providing the unique targets for synthetic lethal interactions. However, the heterogeneity and complexity among different types of cancer make it extremely challenging to universally find the effective synthetic lethal targets. Researchers have described a strategy for identifying highly permeable and actionable genetic interaction core networks called Schematic.

In order to detect which genes are lost and have a synthetic lethal relationship with existing gene mutations driven by cancer (such as TP53, KRAS), researchers used a CRISPR knockout library in genome scale to perform high-throughput screening in a group of human cancer cell lines representing different tissue sources and cancer types.

As a result, multiple targets were found to have synthetic lethal interactions with cancer driven genes (such as TP53 and KRAS mutations), including universally applicable pan-cancer targets (such as the synthetic lethal relationship between USP28 and TP53 deletion) and targets related to specific cancer types (such as the dependence of KRAS mutations on specific cell cycle regulatory genes).

Functional validation shows that knockout or inhibition of these targets can significantly inhibit the proliferation of cancer cells, with minimal impact on normal cells. The targets discovered in this study have drug operability, providing a clear direction for precision cancer treatment and broad-spectrum anti-cancer drug development.

ii. CRISPR Detection

1.Title：Synthetic mismatches enable specific CRISPR-Cas12a-based detection of genome-wide SNVs tracked by ARTEMIS

Journal：Cell Reports Methods（IF：4.3）

Original Link：https://doi.org/10.1016/j.crmeth.2024.100912

Single nucleotide variants (SNVs) have significant importance in personalized medicine and genomics research. Although traditional SNV detection methods such as high-throughput sequencing and PCR are widely used, they have problems such as high cost, limited sensitivity, or difficulty in distinguishing individual base changes. Researchers have proposed a detection strategy based on CRISPR/Cas12a, which combines synthetic mismatches with the newly developed ARTEMIS (Allele specific Reporter Tailored for Enhanced Mismatch Identification and Sensing) platform, enabling specific detection of SNVs across the entire genome.

The introduction of synthetic mismatched crRNA significantly improved ability of Cas12a to distinguish the individual nucleotide differences, enabling the accurate differentiation between target sequences and non target sequences with high similarity; The Cas12a system is capable of detecting DNA sequences as low as sub nanomolar concentrations and is applicable to genomic DNA from different backgrounds; Researchers have successfully validated this platform's ability to detect the key pathogenic SNVs in multiple model genes and patient samples. Compared to traditional sequencing techniques, the CRISPR/Cas12a method has higher time-efficiency and cost-effectiveness. These results provide insights for the rational crRNA design of high fidelity CRISPRdx, supporting the personalized and cost-effective medical solutions in tumor diagnosis.

2.Title：A novel single-tube LAMP-CRISPR/Cas12b method for rapid and visual detection of zoonotic Toxoplasma gondii in the environment

Journal：Infectious Diseases of Poverty（IF：4.8）

Original Link：https://doi.org/10.1186/s40249-024-01266-5

Toxoplasma gondii is an important zoonotic pathogen that can cause serious health problems in both humans and animals. Rapid and sensitive detection of the presence of the pathogen in the environment is of great significance for public health and environmental monitoring. However, existing methods such as PCR are sensitive but time-consuming and require complex equipment, making them difficult to apply in resource limited areas. Researchers have developed a novel detection method based on single tube LAMP-CRISPR/Cas12b, which can quickly, sensitively, and intuitively detect T. in the environment gondii.

This method targets Toxoplasma gondii B1 gene and first amplifies it using LAMP guided by single guide RNA (sgRNA). Then, it recognizes the amplified target gene and activates trans-cleavage, cleaving nearby single stranded DNA (sgRNA) reporter genes. Fluorescence detection was performed using 6-carboxyfluorescein (FAM) -12N-black hole quencher-1 (BHQ1) reporter gene, while fluorescein isothiocyanate (FITC) -12N-biotin can be visually detected on lateral flow test strips.

The LAMP-CRISPR/Cas12b method exhibits high specificity and broad-spectrum detection ability, successfully identifying 9 Toxoplasma gondii genotypes and distinguishing them from 11 other parasites. Sensitivity tests at the molecular (plasmid) and practical (oocyst) levels showed the detection limits of 10 copies/μ L and 0.1 oocyst, respectively. When applied to 112 environmental samples (soil, water, and cat feces), the method demonstrated 100% sensitivity and accurately reflected known infection rates. This LAMP-CRISPR/Cas12b single tube method provides a powerful and innovative approach for monitoring zoonotic toxoplasmosis in environmental samples, which is of great significance for public health monitoring.

iii. Other CRISPR-Related Research

1. Title：High-accuracy crRNA array assembly strategy for multiplex CRISPR

Journal：Molecular Therapy: Nucleic Acid（IF：6.5）

Original Link：https://doi.org/10.1016/j.omtn.2024.102428

CRISPR/Cas9 technology is a powerful gene editing tool that can efficiently and accurately target the genome for modifications. In multiplex CRISPR, simultaneous editing of multiple target sites is an important method to improve the research efficiency for wider application. However, the existing multiplex CRISPR systems often face several problems, such as complex crRNA design, high synthesis costs, and inaccurate guide sequences, which limit their widespread application in genome editing.

Therefore, researchers have proposed a novel, highly accurate, low-cost, and time-saving CRISPR array assembly strategy. Using this strategy, researchers efficiently assembled 12 CRISPR RNAs (crRNAs) (for AsCas12a) and 15 crRNAs (for RfxCas13d) in one reaction. The CRISPR array driven by the Pol II promoter exhibits a different expression pattern than the CRISPR array driven by the Pol III promoter, which can be used to achieve a specific distribution of CRISPR intensity. Subsequently, researchers designed and validated an improved method for expressing long CRISPR arrays. 

This study provides a flexible and powerful tool for easily implementing multiple CRISPR on DNA and RNA, which can help dissect the sophisticated cellular networks and achieve the future realization of multi-target gene therapy. This innovation provides a new tool for the application of multi gene editing technology, with broad potential for biological research and clinical treatment.

2. Title：Construction of a recombinant African swine fever virus with firefly luciferase and eGFP reporter genes and its application in high-throughput antiviral drug screening

Journal：Antiviral Research（IF：4.5）

Original Link：https://doi.org/ 10.1016/j.antiviral.2024.106058

African swine fever (ASF) is a highly lethal contact contagious disease of pigs caused by African swine fever virus (ASFV), primarily infecting domestic pigs and wild boars, with a mortality rate of up to 100%. Currently, there are no commercially available vaccines or drugs that are safe and effective for ASFV. The researchers continuously passaged the ASFV 0428C strain in Vero cells, and the adapted strain ASFV exhibited the efficient replication in Vero cells. Using the adapted ASFV strain as the parent virus, a recombinant ASFV variant strain (rASFV-FLuc-eGFP) was constructed using CRISPR/Cas9 gene editing technology. The rASFV-Fluc-eGFP gene is stable and can effectively infect porcine alveolar macrophages (PAM) and Vero cells, while expressing both Fluc and eGFP simultaneously. This study provides a tool for investigating the infection and pathogenic mechanisms of ASFV, screening essential host genes, and antiviral drugs.

In addition, a high-throughput antiviral drug screening model based on rASFV-FLuc-eGFP was established for passaged cells. 218 compounds were screened from the FDA-approved compound library, and 5 candidate compounds with significant inhibitory effects in Vero cells were identified. Furthermore, its inhibitory effect on ASFV was validated in Vero and PAM cells, and salvianolic acid C (SAC) was identified, which exhibited the inhibitory effects in both cell types and had good safety. SAC is a candidate drug for the prevention and treatment of ASFV, with good application prospects. 

This study successfully established an efficient and sensitive drug screening platform for anti-virus of African swine fever by combining cell culture, infection, reporter gene monitoring, and high-throughput drug screening. In addition, this reporter gene based screening platform can be extended for research on other viruses and drug screening, and has broad potential application value.

II. Industry News

1. The Medical Research Council in the UK has launched two Centers of Research Excellence (CoRE), including the Centre for Therapeutic Genomics, which have received the support of up to £ 50 million over the past 14 years. The center will collaborate with the Innovative Genomics Institute to develop the extensible, artificial intelligence-driven methods for reprogramming gene therapies for incurable genetic diseases, with the aim of reducing costs and accelerating regulatory approvals. The initial targets include blood, eye, and brain diseases.

 
News Link：https://www.ukri.org/news/mrc-launches-two-50m-centres-for-cutting-edge-gene-therapies/

2. ProQR Therapeutics announced an expansion of its partnership with RSRT for Rett Syndrome, receiving $9.1 million in funding to support the advancement of AX-2402 (an Axiomer RNA editing therapy targeting the MECP2 R270X mutation) into clinical trials. This platform has the potential to address various Rett mutations that affect a wide range of patient populations. This collaboration aims to accelerate the transformative therapies for this severe neurodevelopmental disorder, which has a significant unmet need.

News Link：https://www.proqr.com/press-releases/proqr-therapeutics-announces-81-million-in-new-funding-from-rett-syndrome-research-trust-to-expand-rna-editing-collaboration

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.

Recent Blogs

1. [Literature Review] CRISPR gene editing combined with magnetocaloric activation: A new strategy to assist precision medicine in cancer

2. [Weekly News] Multiplexed CRISPR Arrayed Libraries: Advancing Whole-Genome Targeting for Knockout, Activation, and Silencing Research

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

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