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:：BES-Designer: A Web Tool to Design Guide RNAs for Base Editing to Simplify Library

Journal：Interdisciplinary Sciences-Computational LIfe Sciences（Impact Factor：3.9）

Original Link：https://doi.org/10.1007/s12539-024-00663-6

CRISPR/Cas base editors allow for precise single-nucleotide conversions without causing double-strand breaks. This technology is widely used in gene therapy, gene function studies, and other applications. While there are many tools for designing gRNAs, creating simplified base editing libraries for gRNA screening remains challenging, especially when dealing with libraries that include multiple adjacent protospacer adjacent motifs (PAMs).

To tackle this, researchers have introduced BES-Designer, an innovative web tool for designing gRNAs specifically for base editors. This tool is designed to streamline the creation of base editing libraries. BES-Designer uses target sequence simplification rules that help researchers more efficiently narrow down their experimental targets.

By optimizing the selection of target sequences for various PAMs and editing types, BES-Designer enhances the design of gRNAs for CRISPR-Cas base editing. Experimental results show that the tool can improve efficiency by up to 30% in base editing libraries.

2.Title：TMEM106B-mediated SARS-CoV-2 infection allows for robust ACE2-independent infection in vitro but not in vivo

Journal：Cell Rep.（Impact Factor：7.5）

Original Link：https://doi.org/10.1016/j.celrep.2024.114921

SARS-CoV-2 primarily enters host cells through the ACE2 receptor on the cell surface. However, recent studies suggest that some cells can still be infected by the virus even without ACE2. This points to a potential ACE2-independent infection mechanism.

To explore this, researchers used a SARS-CoV-2 mouse-adapted strain (SARS-CoV-2 MA1) with the E484D spike mutation and conducted a whole-genome CRISPR/Cas9 knockout screening to identify alternative entry pathways. They focused on TMEM106B, a transmembrane protein previously linked to neurodegenerative diseases. The researchers hypothesized that TMEM106B could play a role in helping SARS-CoV-2 infect cells in the absence of ACE2.

The researchers performed experiments using both in vitro cell models and mouse models. In the in vitro studies, they edited cells to express TMEM106B without ACE2 and then tested the cells' ability to be infected by SARS-CoV-2. The results showed that TMEM106B could indeed facilitate SARS-CoV-2 infection, even in cells lacking ACE2. These cells were sensitive to the virus, which was able to enter and replicate inside them.

However, in mouse models, the ACE2-independent infection mechanism was not as prominent. This study suggests an additional potential pathway for SARS-CoV-2 infection, while also highlighting the differences between in vitro and in vivo findings. While ACE2 remains the primary entry route for the virus, the discovery of TMEM106B opens up new directions for future research.

ii. CRISPR Knockout Cell Lines

1. Title：Expanding the cell quantity of CRISPR/Cas9 gene editing by continuous microfluidic electroporation chip

Journal：Bioelectrochemistry（Impact Factor:4.8）

Original Link：https://doi.org/10.1016/j.bioelechem.2024.108840

CRISPR/Cas9 technology has great potential for genome editing, but it faces challenges in large-scale cell editing. Specifically, it's difficult to maintain high editing efficiency while minimizing cell damage. Traditional electroporation methods can deliver CRISPR/Cas9 to cells efficiently, but they struggle to balance high cell survival rates with processing large numbers of cells at once.

To address this, the researchers developed the LaViE-Chip, a continuous microfluidic electroporation device. This chip uses multiple narrow microfluidic channels arranged in parallel. It achieves a good balance between cell flow and electric field uniformity with just two simple external electrodes. The design also isolates the harmful effects of the electrodes, protecting the target cells. Additionally, the microfluidic channels are curved to control the fluid dynamics and make the target cells rotate, which boosts both transfection efficiency and cell viability.

With these improvements, LaViE-Chip achieved 71.06% electroporation efficiency, 84.3% cell viability, and a processing speed of 107 cells per minute. This method provides high cell survival, efficient editing, and continuous operation, making it ideal for large-scale cell editing. The researchers also successfully demonstrated continuous CRISPR gene editing via electroporation for the first time. This breakthrough opens up exciting possibilities for large-scale gene editing and personalized medicine in the future.

2. Title：Generation of CRISPR/Cas9 modified human iPSC line with correction of heterozygous mutation in exon 6 of the CaSR gene

Journal：Human Cell（Impact Factor:3.4）

Original Link：https://doi.org/10.1007/s13577-024-01135-1

The CaSR gene encodes the calcium-sensing receptor (CaSR) protein, which is crucial for regulating calcium balance in the body. Mutations in the CaSR gene are linked to conditions like familial hypocalciuric hypercalcemia (FHH). To verify this, researchers used CRISPR/Cas9 gene editing to correct these mutations. They edited human induced pluripotent stem cells (iPSCs) to create cell lines without the mutations.

The results were promising, the researchers successfully corrected a heterozygous mutation in exon 6 of the CaSR gene in iPSCs. They created cell lines with a normal CaSR gene sequence. The correction was accurate, and in the edited iPSCs, CaSR gene expression and calcium-sensing function were restored. This shows that the corrected cells can express functional CaSR protein, confirming the success of the gene editing.

This study offers a reliable model for exploring calcium homeostasis and developing personalized treatments for related diseases, as well as new insights for future research on gene mutation-based personalized therapies.

iii. CRISPR Detection

1. Title：Characterization of RNA editing and gene therapy with a compact CRISPR-Cas13 in the retina

Journal：Proc Natl Acad Sci U S A.（Impact Factor：9.4）

Original Link：https://doi.org/10.1073/pnas.2408345121

The CRISPR-Cas13 system is an RNA-targeting gene editing tool that avoids the risks of making permanent changes to the genome. Because retinal cells may be sensitive to permanent gene edits, researchers have turned to Cas13 RNA editing as a safer alternative for non-permanent gene therapy.

The goal was to develop a compact CRISPR-Cas13 system that could be easily delivered to the retina, and test its ability to edit specific mutated genes in retinal cells.

The results were promising. The compact Cas13 system showed efficient RNA editing in an in vitro retinal cell model. It successfully targeted and corrected specific mutated RNA sequences with high accuracy and specificity, demonstrating its potential for treating retinal diseases.

The compact design of the Cas13 system makes it easier to deliver, even to small target cells that are difficult to treat with other gene therapies. This non-permanent RNA editing approach offers a safer strategy for retinal disease treatment. With its easy delivery and strong biocompatibility, the system shows great promise for future use in gene therapy.

2. Title：Analyte-induced hindrance in the RCA-assisted CRISPR/Cas12a system for homogeneous protein assays

Journal：Analytica Chimica Acta（Impact Factor：5.7）

Original Link：https://doi.org/10.1016/j.aca.2024.343294

Enzyme-linked immunosorbent assays (ELISA) and other heterogeneous detection methods are crucial in in vitro diagnostics. However, their simplicity, sensitivity, and accuracy are often limited by multiple washing and incubation steps, as well as limited amplification techniques. To overcome these challenges, researchers have developed a new method that combines rolling circle amplification (RCA) with the CRISPR/Cas12a system. This approach enables simple, highly sensitive, and homogeneous protein detection.

In this method, streptavidin (SA) and digoxin antibody (anti-Dig) are used as model targets. Small molecule-modified primers specifically recognize the target proteins, blocking the RCA process. This prevents the activation of Cas12a's  trans-cleavage activity, leading to a decrease in fluorescence intensity.

The platform showed exceptional performance, with high sensitivity, strong specificity, and great potential for use in complex samples. By expanding the recognition elements, this system could become a versatile, multifunctional tool for clinical diagnostics. It also offers a new approach for quantifying ultra-low concentration disease biomarkers in clinical practice.

iv. Other CRISPR-Related Research

1. Title：DTMP-Prime: A Deep Transformer-based Model for Predicting Prime Editing Efficiency and PegRNA Activity

Journal：Molecular Therapy: Nucleic Acid（Impact Factor：6.5）

Original Link：https://doi.org/10.1016/j.omtn.2024.102370

Prime Editing (PE) is a CRISPR-based gene-editing tool with great potential for correcting genetic mutations. However, to achieve high editing efficiency, the Prime Editing Guide RNAs (PegRNAs) need to be optimized. Researchers from Iran and Germany have developed a deep learning model called DTMP-Prime to improve predictions of PE efficiency and PegRNA activity. This tool helps identify the best combinations of PegRNAs and nick gRNAs (ngRNAs) and accurately predicts the efficiency and results of PE experiments.

DTMP-Prime also shows promise in predicting off-target sites in CRISPR experiments. Evaluations using Pearson and Spearman correlation coefficients demonstrate that DTMP-Prime outperforms other leading models in predicting the success of PE experiments. This tool plays an important role in supporting gene-editing research, aiding to advance the use of Prime Editing in medical research and clinical applications.

2. Title：RegⅢγ promotes the proliferation, and inhibits inflammation response of macrophages by Akt, STAT3 and NF-κB pathways

Journal：International Immunopharmacology（Impact Factor：4.8）

Original Link：https://doi.org/10.1016/j.intimp.2024.113442

Regenerating gene family protein III gamma (RegⅢγ) is a secreted protein that is typically expressed in intestinal epithelial cells. It plays an important role in tissue damage and inflammation. However, it is still unclear whether RegⅢγ in the liver contributes to liver regeneration (LR).

To investigate this, researchers used microarray analysis, qRT-PCR, and immunofluorescence staining to examine the expression and localization of RegⅢγ during LR. They then created RegⅢγ-deficient and overexpressing RAW264.7 cells using CRISPR/Cas9 and lentiviral infection.

The researchers conducted several assays to study the role of RegⅢγ in cell proliferation and inflammation. These included MTT assays, flow cytometry, EdU assays, transwell migration, neutral red phagocytosis, and NO production experiments. They also used immunoprecipitation and Western blotting to explore RegⅢγ's regulatory mechanisms.

The results revealed that RegⅢγ expression in Kupffer cells changes significantly during LR. Overexpression of RegⅢγ enhanced RAW264.7 cell viability, proliferation, phagocytosis, and migration. Conversely, deleting RegⅢγ reversed these effects. Additionally, overexpression of RegⅢγ increased the expression of HO-1 and IL-10, while RegⅢγ deletion led to higher NO production and increased levels of p-Akt, p-STAT3, p-p65, and TNF-α.

In conclusion, during the early stages of liver regeneration, RegⅢγ likely promotes LR by stimulating macrophage proliferation and reducing inflammation through the Akt, STAT3, and NF-κB pathways. This discovery not only deepens the understanding of macrophage function, but also offers new potential strategies for treating inflammatory diseases.

II.Industry News

1.Allogene Therapeutics recently announced that its CAR-T candidate, ALLO-316, has received the FDA's Regenerative Medicine Advanced Therapy (RMAT) designation. ALLO-316 is based on Transcription Activator-Like Effector Nuclease (TALEN) gene editing technology. The RMAT designation is given to products that show potential to meet serious, unmet medical needs. It is intended to speed up the development and regulatory review of these products. ALLO-316 is being developed for the treatment of CD70-positive advanced or metastatic renal cell carcinoma (RCC).

News Link： https://ir.allogene.com/news-releases/news-release-details/allogene-therapeutics-receives-fda-regenerative-medicine

2.At the recent ESGCT conference, Precision BioSciences highlighted the impressive efficiency of its ARCUS nuclease for gene insertion. Using an AAV6 vector, they achieved over 85% homologous directed repair (HDR) integration in both T cells and liver cells. The targeted insertion is made possible by ARCUS's unique staggered 3' overhang cutting. This approach not only supports base editing and deletions but also enables large-scale genomic replacements.

News Link： https://precisionbiosciences.com/wp-content/uploads/2024/10/ESGCT-2024-poster-final42.pdf

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] Gene Knockout of ERAP1 Provides New Insights for Optimizing Tumor Immunotherapy
2.[Weekly News]CRISPR/Cas9 Gene Knockout: A New Strategy to Overcome Immune Therapy Resistance in Neuroblastoma
3.[Weekly News] CRISPR/Cas9 Screening Unlocks Secrets of Stem Cell Power

Follow us on social media

Contact us

+ 833-226-3234 (USA Toll-free)

+1-224-345-1927 (USA)

info@editxor.com