CMN Weekly (26 April 2024) - Your Weekly CRISPR Medicine News

Top picks

• In a not-yet-peer-reviewed paper, American researchers have used large language models trained on vast biological data - over one million CRISPR operons - to generate an algorithm, OpenCRISPR-1, that can design diverse CRISPR-Cas proteins capable of precision human genome editing. Several generated gene editors show comparable or improved activity and specificity relative to SpCas9, while 400 mutations away in sequence. OpenCRISPR-1, also compatible with base editing, has been made publicly available to encourage wide, ethical use in various fields. The achievement is also featured in an article in the New York Times.
• The phase 3 clinical study results that led to the recent approval of the world's first CRISPR gene-editing therapy, Casgevy from Vertex, have now been published in two separate papers. One study found that Casgevy eliminated vaso-occlusive crises in 97% of patients with sickle cell disease for 12 months or more. The other study found that treatment resulted in transfusion independence in 91% of patients with transfusion-dependent β-thalassemia.

Research

• American researchers have refined CRISPR base-editing for disease-linked variant analysis with a new tool, BEAN, that integrates experimental and computational data, enhancing accuracy in variant effect quantification. BEAN effectively identified genes impacting LDL uptake and accurately assessed LDLR variant pathogenicity, showing a strong correlation with clinical data.
• Italian researchers have discovered CoCas9, a nuclease from an uncultivated Collinsella species, by interrogating 17,173 type II CRISPR-Cas loci from metagenome-assembled genomes primarily sourced from human microbiomes. CoCas9 stands out for its strong activity, high fidelity, and compact size (1004 amino acids) and has demonstrated efficient in vivo gene editing in the mouse retina after AAV delivery.
• In a novel approach named CellEDIT, German researchers have streamlined glycoengineering in CHO cells by intranuclear delivery of CRISPR components using FluidFM technology. This technique enabled the rapid generation of triple knockout CHO-K1 cell lines targeting BAX, DHFR, and FUT8 genes, bypassing traditional methods like limiting dilution and cell sorting. The new method is promising for improving monoclonal antibody production in CHO cells.
• German researchers have used an inducible CRISPRi human induced pluripotent stem cell (hiPSC) line to silence telomerase (TERT) expression, thereby generating hiPSCs and hiPSC-derived cardiomyocytes with long and short telomeres. Reduced telomerase activity and shorter telomere lengths of hiPSCs induced global transcriptomic changes associated with cardiac developmental pathways.
• Researchers in the UK have used genome-wide CRISPR screens to identify critical pathways contributing to drug resistance in EGFR mutant lung cancer, particularly involving the Hippo pathway. Targeting both Hippo signalling and EGFR simultaneously proves highly effective in treating EGFR mutant cells and patient-derived organoids.
• American researchers have shown that site-specific RNA breaks generated with type III CRISPR complexes are repaired in human cells, and this repair can be used to restore gene function through programmable deletions in human transcripts. The work establishes a technology for precise RNA manipulation with potential therapeutic applications.
• Chinese researchers have used CRISPR base-editing to generate a KCNQ1 (c.1032 + 2 T > C) mutant human embryonic stem cell line. The WAe009-A-1D cell line maintains stem cells' morphology, pluripotency, and normal karyotype and can differentiate into all three germ layers in vivo.
• Researchers in the Netherlands have enhanced CRISPR-Cas systems, creating PAM-relaxed variants SpRYCas9 and LbCas12a to expand genome editing targets. Using catalytically inactive (dead) forms fused with a photoactivatable protein, they tracked these in E. coli and showed that LbdCas12a variants locate DNA targets faster than SpydCas9, especially with targeting RNA guides.
• In a study aimed at treating β-thalassemia and sickle cell disease, researchers have successfully used CRISPR-Cas9 to edit the BCL11A enhancer in cord blood-derived hematopoietic stem and progenitor cells (CB-HSPCs) to reactivate fetal haemoglobin (HbF) in erythroblasts. This editing achieved high indel frequencies (74–80%) and a significant 93% reduction in BCL11A RNA levels, resulting in a marked increase in γ/β-globin transcript ratios (from 11 to 77-fold) and HbF levels (from 34% to 44%).
• Chinese researchers have created a photoactivatable crRNA by combining CRISPR/Cpf1 with optogenetics and a reduction-responsive motif. This system enables precise genome editing when exposed to light and can be deactivated with a reducing agent.

Industry

• SNIPR Biome has received $5.48 million from CARB-X to further advance SNIPR001 into a Phase 1b/2a clinical trial in 24 blood cancer patients across Europe and the United States. SNIPR001 is a novel, orally administered antibiotic designed to target difficult-to-treat bacterial infections precisely.has presented preclinical data demonstrating over 90% reduction in viral shedding in a rabbit model of herpes simplex virus-1 keratitis (HSV-1 Keratitis) after in vivo injection of EBT-104. EBT-104 is a CRISPR-Cas9-mediated gene editing therapeutic candidate employing single all-in-one AAV8(Y733F) and AAV9 vector delivery of SaCas9 and paired gRNAs.
• ProQR Therapeutics announces it has successfully defended an opposition filed against a key patent for its ADAR-mediated RNA editing platform, Axiomer. The patent dispute concerns an antisense oligonucleotide (AON) capable of forming a double-stranded complex with a target RNA sequence for the deamination of a target adenosine in the target RNA sequence by an ADAR enzyme present in the cell.

Detection

• A new microplate assay for small molecules combines the advantages of structure-switchable aptamer-mediated signal conversion and CRISPR-Cas12a-based signal amplification. The assay was demonstrated by detecting two highly toxic pollutants, aflatoxin B1 (AFB1) and cadmium ion (Cd2+), and exhibited good selectivity and high sensitivity, with detection limits of 31 pM and 3.9 nM, respectively.
• One-pot-RPA-CRISPR/Cas12a is a novel nucleic acid testing method that combines RPA with CRISPR molecular diagnostic technology. It enables the simultaneous detection of 12 common respiratory pathogens, including six bacteria and six viruses. RPA and CRISPR-Cas12a reactions are separated by paraffin, providing an independent platform for RPA reactions to generate sufficient target products before being mixed with the CRISPR/Cas12a system. Results can be visually observed under LED blue light.
• A novel ultrasensitive and reliable biosensor for circulating tumour DNA (ctDNA) utilises exponential amplification reaction (EXPAR)-assisted CRISPR/Cas12a-mediated ratiometric dual-signal electrochemical detection. The method demonstrated satisfactory ctDNA detection in complex human serum, highlighting its potential for cancer diagnosis.
• PLACID (Paper-based LAMP-CRISPR Integrated Diagnostics) is a new paper-based nucleic acid testing platform that merges Loop-mediated isothermal amplification (LAMP) with CRISPR-Cas12a technology for simplified, on-paper amplification and detection. The system is operated via a smartphone-controlled IR heating chamber and achieves high specificity and sensitivity with a detection limit of 50 copies/μL of SARS-CoV-2 RNA or E. coli.
• A new rapid and efficient testing platform for detecting the foodborne pathogen E. coli O157:H7 is based on CRISPR-Cas12a-derived fluorescence and lateral flow chromatography. The assay can be completed within 45 min.

Reviews

• Base Editors-Mediated Gene Therapy in Hematopoietic Stem Cells for Hematologic Diseases. This review summarises the development and recent progress of DNA base editors, discusses their applications in HSC gene therapy, and highlights the prospects and challenges of future clinical stem cell therapies.
• Advances in miniature CRISPR-Cas proteins and their applications in gene editing. This review systematically evaluates the latest research progress on several miniature CRISPR-Cas proteins (Cas12f, Cas12j, Cas12k, and Cas12m) and their practical applications in gene editing.
• Advances, challenges, and opportunities for food safety analysis in the isothermal nucleic acid amplification/CRISPR-Cas12a era. The review summarises various detection methodologies by integrating isothermal nucleic acid amplification and CRISPR-Cas12a technology for diverse food safety concerns, including pathogenic bacteria, viruses, mycotoxins, food adulteration, and genetically modified foods.
• CRISPR screens in mechanism and target discovery for AML. This review presents an overview of recent progress in developing CRISPR-based screens for the mechanism and target identification in AML and discusses the challenges and possible solutions in this rapidly growing field.