CMN Weekly (19 April 2024) - Your Weekly CRISPR Medicine News

Top picks

• CRISPR-Cas is challenged by a new programmable DNA and RNA targeting technology discovered in short prokaryotic Argonautes (pAgos) from Novosphingopyxis baekryungensis and Cupriavidus metallidurans. These pAgos, in partnership with SPARDA nucleases, enable RNA-guided systems that trigger indiscriminate DNA and RNA cleavage, leading to cell dormancy or death. This mechanism, discovered by Russian scientists, extends our knowledge of prokaryotic immune responses, providing targeted population protection that parallels yet differs from CRISPR-Cas systems.
• In a one-hour webcast, Jennifer Doudna discusses the future of CRISPR and what's ahead for genome editing. The talk is part of the NIH Director's Wednesday Afternoon Lecture Series, colloquially known as WALS, the highest-profile lecture program at the NIH. Among other topics, the Nobel Laureate discusses the recent FDA approval of the first CRISPR therapy and ongoing research to expand access and reduce the costs of CRISPR medicines.

Research

• In vivo CRISPR-Cas9 screening in a murine lung cancer model has revealed that oncogenic KRAS promotes immune evasion in lung cancer by upregulating COX-2, which via prostaglandin E2, resists immune checkpoint blockade. Targeting the COX-2/PGE2 pathway remodelled the tumour microenvironment, enhancing immunotherapy efficacy by attracting cytotoxic CD8+ T cells. These findings suggest combining COX-2 inhibitors with KRASG12C inhibitors or an immune checkpoint blockade could benefit KRAS-mutant lung cancer treatment.
• German researchers have engineered a new CRISPR-Cas13d variant, Cas13d-NCS, that enables efficient cytosolic RNA targeting in mammalian cells. This variant improves the transfer of crRNAs from the nucleus to the cytosol, significantly enhancing the degradation of mRNA and viral RNA, including complete inhibition of SARS-CoV-2 replication.
• Researchers have developed dual-functionalised polymers with boronate and lipoic acid that can efficiently deliver proteins, including Cas9 ribonucleoprotein, in vivo into skin tissue. This method overcomes the challenge of membrane permeability, facilitating endosomal escape and intracellular release. In a mouse model of psoriasis, the optimal polymer disrupted NLRP3 inflammasomes, reducing inflammation and showcasing the potential for targeted CRISPR-Cas9 gene therapy in inflammatory skin disorders.
• A new study introduces an acoustothermal transfection method combining acoustic and thermal effects to enhance cellular permeability. Achieving nearly 89.6% efficiency, this technique allows high-throughput transfection of T cells and stem cells while preserving viability. Successfully used in mesenchymal stem cells, it targeted and reduced cerebral ischemia in mice, promising for future gene therapies.
• New findings highlight a secondary conformational checkpoint in CRISPR-Cas9 that significantly enhances its accuracy in gene editing. Using dynamic simulations, researchers have discovered that the presence of base mismatches in DNA triggers alterations in the HNH nuclease domain of Cas9, thereby hindering its conformational activation.
• A new study showcases consistent long-term survival of pig-to-non-human primate (NHP) kidney xenografts using genetically modified pigs, achieved with FDA-approved immunosuppression methods. The pigs were modified using CRISPR-Cas gene editing to enhance compatibility and reduce rejection risks. This marked a significant step towards applying these methods in clinical xenotransplantation to alleviate organ shortages.
• American researchers have developed a novel CRISPR-based platform to identify and characterise essential nutrient transporters that support cancer cell proliferation. Employing a CRISPR interference and activation (CRISPRi/a) screening platform, the researchers systematically identified crucial nutrient transporters that support cancer cell proliferation.
• In vivo CRISPR perturbation of the Rheb gene has revealed its crucial role in enhancing drug-induced behaviour and diminishing natural reward consumption. This study highlights the nucleus accumbens as central to drug disruption of natural rewards, being altered by chronic drug exposure.
• Building on the CRISPR-based RNA-United Interacting System (CRUIS), researchers have developed an enhanced version, eCRUIS, utilising dCas13d and TurboID ligase. This innovation allows for rapid labelling of RNA-binding proteins on target RNA within 30 minutes, suitable for both in vitro and in vivo applications. Validation with exogenous RNA bait assays confirmed eCRUIS's efficacy in quickly identifying RNA-protein interactions, broadening its application in molecular biology research.
• Russian scientists have used CRISPR-Cas9 to create a TDP1 knockout in HEK293A cells, revealing through transcriptomic analysis that TDP1 influences various biological processes. These include cell adhesion, spermatogenesis, mitochondrial function, neurodegeneration, cytokine responses, and the MAPK signalling pathway, suggesting TDP1's role extends beyond DNA repair.

Industry

• Precision BioSciences has regained control of three programs using its proprietary ARCUS in vivo gene editing platform licensed to Prevail Therapeutics in 2021. The non-disclosed programs will now be prepared for GLP toxicology studies, followed by potential IND and clinical trial application submissions

Detection

• Cocaine and methamphetamine are now detectable in sweat within one hour with a new point-of-care CRISPR-Cas12a-based system. The sensor employs aptamer and magnetic separation technology, achieving a detection limit as low as 0.1 ng/mL.
• Chinese scientists have developed a single-step CRISPR-based diagnostic platform, SCOPE (Streamlined CRISPR On Pod Evaluation platform), for field-deployable ultrasensitive monkeypox virus (MPXV) detection in resource-limited settings. SCOPE can detect as low as 0.5 copies/µL (2.5 copies/reaction) of MPXV within 15 min from the sample input to the answer.
• Researchers in China have developed a label-free electrochemiluminescence (ECL), Cas12a-based biosensor for HPV-16 DNA by encapsulating carbon dots in DNA hydrogel. Activation by target DNA via Cas12a enabled rapid and simple target detection, achieving a sensitivity of 0.63 pM directly from human serum samples in about 60 minutes.
• A new sensitive fluorescence biosensor to detect the H1N1 virus employs ligation-transcription and CRISPR-Cas13a-assisted cascade amplification strategies. The biosensor sensitively and specifically monitored H1N1 viral RNA levels down to 3.23 pM and showed good linearity when H1N1 RNA concentrations were 100 pM-1 µM.

Reviews

• Evolution of Prime Editing Systems: Move Forward to the Treatment of Hereditary Diseases. This review observes the development of the platform and discusses the candidate proteins for efficiency-enhancing, main delivery methods and current applications of prime editing.
• CRISPR-Cas gene knockouts to optimise engineered T cells for cancer immunotherapy. This review revisits establishing the dysfunctional profile of T cells before delving into a comprehensive summary of recent CRISPR-gene invalidations, with each invalidation contributing to the enhancement of engineered T cells' antitumor capacities.
• dCas9 Tells Tales: Probing Gene Function and Transcription Regulation in Cancer. This review delves into the advancements in Cas9 protein engineering, specifically the generation of various dCas9 tools that have significantly enhanced the capability and versatility of CRISPR-based technology.
• CRISPR-Cas9 applications in T cells and adoptive T cell therapies. This review provides a comprehensive summary of the role of CRISPR–Cas9 in T cells and its applications in preclinical and clinical studies for T cell-based therapies.

Perspectives

• A feature in Nature Biotechnology highlights the competitive IP landscape in prime editing, including the prime editing patents secured by Liu's lab. These patents cover methods for site-specific DNA modifications using a combination of Cas9 and reverse transcriptase. These patents set a precedent that may require newer technologies to navigate carefully to avoid infringement, influencing strategic decisions like mergers and licensing in the biotech industry to secure access to crucial patented technologies. A list of current prime editing patents is available.
• A feature in Nature Reviews Drug Discovery describes the emergence of RNA-editing therapies. Recently, at least three RNA-editing drugs have reached clinical trials or received approval to begin trials. These therapies offer the potential for precise genetic modifications without permanent changes to the DNA, providing a flexible approach for treating a variety of conditions, including cancer, pain, and high cholesterol.

Ooh, aah, wow

• CRISPR has been used to edit and clone cats, thereby achieving offspring with light-coloured mackerel tabby coats, which are highly demanded. Using a pair of gRNAs, the Chinese scientists deleted exon 17 of KIT (a key gene controlling melanoblast differentiation and melanogenesis) in somatic cells isolated from two different Li Hua feline foetuses. Nuclei from edited cells were then used as donors for cloning, achieving 1.5% (2/131) kittens with an obvious darkness reduction in the mackerel tabby coat.

News from CRISPR Medicine News

• Hopefully, you have already signed up for the CRISPR Medicine Conference 2024, which we proudly present next week in Copenhagen. More than 550 attendees will gather to listen to over 50 talks and watch 100+ posters. We are looking forward to seeing you!