CMN Weekly (10 December 2021) - Your Weekly CRISPR Medicine News

Top Picks

• Chinese researchers have developed a CRISPR-Cas9 prodrug nanosystem (NanoProCas9), which combines the targeted delivery and the conditional activation of CRISPR-Cas9 for the precision therapy of inflammatory bowel disease. After systemic administration of NanoProCas9, the destabilised Cas9 is activated only in inflammatory lesions by pathological ROS signals.
• David Liu and colleagues have developed a new CRISPR-based gene editing technique called twin prime editing (twinPE) that could be a new and safer approach to gene therapy. The technique makes two adjacent prime edits to introduce larger sequences of DNA at specific locations in the genome with few unwanted by-products.
• Adeno-associated virus (AAV) capsid variants with increased tropism for the brain and targeted away from the liver have been engineered by researchers from Caltech. The tropism and the ability to cross the blood-brain barrier was demonstrated in mouse and marmoset and enabled robust, non-invasive gene delivery to the brain after intravenous administration.

Research

• FiCAT (find and cut-and-transfer) is a new method for targeted integration of large DNA fragments in mammalian genomes proposed by Spanish researchers. The technique combines CRISPR-Cas9 as the find module and an engineered piggyBac transposase as the cut-and-transfer module. More efficient versions of FiCAT generated by directed evolution enabled targeted insertion of multi kilobase DNA fragments in mammalian genomes.
• Danish researchers have developed a new comprehensive software tool, CRISPRroots, that combines off-target predictions and RNA sequencing analysis to evaluate gene editing outcomes. Read our interview with the researchers.
• Researchers in the UK have characterised the specificity and sensitivity of an RNA targeting type III CRISPR complex that detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. Furthermore, the researchers demonstrate how the system can be used to detect the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step.
• X-ray crystal structures of the HNH endonuclease domains of two Cas9 proteins from G. stearothermophilus and S. pyogenes reveal that the core of HNH is conserved despite low sequence similarity. However, the authors found other significant differences in the dynamics of the two nucleases.
• Italian researchers have developed a hyperthermophilic Cas9-based genome-editing tool, active up to 65°C, for accelerated editing of the genome of T. thermophilus. The researchers used the method to characterise the bacteria's arsenite detoxification system and develop a sensitive whole-cell bioreporter for arsenic ions.
• The compact Cas9 from Staphylococcus chromogenes is shown to recognise a simple NNGR PAM sequence, representing the most relaxed PAM preference of compact Cas9s to date. The Chinese researchers also demonstrate that SchCas9 enables efficient genome editing in multiple human cell lines.

Industry

• The German biotechnology company BRAIN Biotech has announced the discovery of a new Class 2 CRISPR nuclease, BRAIN-Metagenome-Cas 01 (BMC01). BMC01 is one of around 2.000 related nucleases that the company has identified in-silico. BRAIN Biotech has filed IP protection for BMC01 and 15 other nucleases.
• The genetic medicines company Metagenomi has announced the presentation of new preclinical data for their in vivo and ex vivo type V Cas gene delivery system using lipid nanoparticles designed for immunotherapy approaches to treat blood cancers. The novel, compact, and hypoimmune gene-editing system will be presented at the 63^rd Annual Meeting and Exposition of the American Society of Hematology (ASH) on December 13.
• Also, at ASH on December 13, Emendo Biotherapeutics will present data from a CRISPR-based ex vivo strategy involving allele-specific excision of the mutant ELANE allele responsible for severe congenital neutropenia. The data will be presented in a poster entitled: "Safe and Efficient Engraftment of CRISPR-based ELANE Mono-Allelic Knocked Out HSCs in Mice: Evidence for a Novel Treatment for ELANE Neutropenia".
• Precision BioSciences has presented preclinical gene editing data for its chronic hepatitis B virus (HBV) program. The company uses its proprietary ARCUS gene-editing technology based on the ARC Nuclease, a fully synthetic enzyme derived from the natural homing endonuclease I-CreI. Results showed that ARCUS efficiently targeted and degraded HBV cccDNA and reduced expression of HBV s-antigen by 77% in HBV-infected primary human hepatocytes.

Detection

• Australian researchers present an aptamer-triggered CRISPR-Cas12a assisted on-fibre immunosensor (CAFI) to detect small proteins. CAFI can measure proteins in low (100 μL) volumes of complex biological samples and is easily adapted to specific proteins by modifying the capture antibody and detection aptamer.
• American researchers describe a highly sensitive immuno-CRISPR assay for CXCL9 detection. The assay uses anti-CXCL9 antibody–DNA barcode conjugates and Cas12a-based trans-cleavage activity of FQ reporter substrates. CXCL9 is markedly elevated in the urine of kidney transplant recipients undergoing acute rejection episodes. In urine samples from 11 kidney transplant recipients, the assay resulted in 100 pct accuracy to clinical CXCL9 determination.
• Researchers in China demonstrate how the CRISPR-Cas system can be applied as a signal enhancer for DNAzyme-based lead ion detection. Lead ions can be detected at 0.48 nM, and the naked eye can visualise detection results with a portable 3D printing device.

Reviews

• A Chinese review gives a comprehensive overview of the power and the promise of CRISPR-Cas9 genome editing for clinical application with gene therapy. The authors present the history, classification and mechanism of CRISPR and its therapeutic applications.
• A review by international researchers looks at engineered CRISPR-Cas systems to detect and control antibiotic-resistant infections. The review also summarises the most recent advances in applying the CRISPR-Cas system for virulence modulation of bacterial infections and combating antimicrobial resistance.

Commentary

• A news & views article in Nature Biotechnology discusses three recent papers that have developed and applied a new CRISPR-based screening method called Repair-seq to investigate the interplay between editing tools and the cellular DNA repair machinery.

Huh, Heh, Wow

• American researchers have developed four real-time detection systems that can spontaneously indicate the presence of active CRISPR-Cas tools for genome editing and gene regulation, including CRISPR-Cas9 nuclease, base editing, prime editing, and CRISPRa in plants. The systems employ fluorescence-based molecular biosensors in transient expression via protoplast transformation, leaf infiltration and stable transformation. The method can help comply with concerns about risks associated with the CRISPR-Cas technology.
• Two other papers take on the debate about the ethics of CRISPR-editing in agriculture. One commentatory discusses whether agroecology and CRISPR can mix. The other examines CRISPR and its regulation in the EU for food business operators relating to the traceability and labelling of food products.