CMN Weekly (20 May 2022) - Your Weekly CRISPR Medicine News

Top picks

• CRISPR gene editing is now possible in cockroaches (Blattella germanica). The Japanese and Spanish researchers used the straightforward and effective "direct parental" CRISPR (DIPA-CRISPR) procedure that involves injecting materials into female adults where eggs are developing rather than into the embryos themselves. The authors believe that the same approach can be used to gene edit more than 90% of insect species.
• Researchers from Rice University, Texas, have achieved large-scale multiplex base- and prime-editing in human cells. The scientists developed a so-called drive-and-process (DAP) CRISPR array architecture that leverages an engineered 75-nt human cysteine tRNA to release individual gRNAs by the cellular endogenous tRNA processing machinery. Up to 31 and 3 loci, respectively, could be base-edited and prime-edited simultaneously, potentially treating polygenic diseases.

Research

• Researchers from Precision BioSciences and Gilead Sciences have described a potential curative approach for chronic Hepatitis B (CHB) using a highly specific engineered ARCUS nuclease (ARCUS-POL) targeting the Hepatitis B virus (HBV) genome. Systemic administration of lipid nanoparticles containing the gene-editing molecules in mice and non-human primates resulted in a significant decrease in surrogate-HBV copy number and high on-target indel frequency.
• Researchers in China describe an advanced system for spatial-controlled RNA editing and precise cancer therapy. The system employs self-uncloaking CRISPR-Cas13a–customised RNA nanococoons (RNCOs-D), featuring tumour-specific recognition and spatial-controlled activation of Cas13a. NCOs-D consists of programmable RNA nanosponges (RNSs) capable of targeted delivery and nanocapsules (NCs) anchored on RNSs for cloaking Cas13a/crRNA ribonucleoprotein activity.
• Researchers from AstraZeneca, Sweden, have developed extracellular vesicles (EVs) containing Cas9 as a novel gene-editing tool. Approximately 25 Cas9 molecules could be loaded per EV, and they achieved gene editing efficiencies of 51% and 6% for the Cre reporter cassette and the PCSK9 gene, respectively, in HEK293 cells.
• Researchers in the USA have used CRISPR-Cas9 to knockout interleukin-1 receptor-associated kinase 3 (IRAK3) in monocyte-derived dendritic cells. This increased IL-12 production upon lipopolysaccharide (LPS) stimulation, which is associated with enhanced antitumor immunity.
• A novel sgRNA design tool, ExsgRNA, takes on-target gRNA-DNA mismatches into consideration. Experiments showed that multiple sgRNAs with three mismatches could achieve 80% of the relative activity of the perfect matched sgRNA and at the same time reduce the number of off-target sites.
• American researchers introduce a pathway model to direct correction of point mutations after CRISPR-Cas12a gene editing. The method utilises templates for homology-directed correction. When used in a cell-free gene-editing system, the authors could directly correct four unique point mutations, including two unique nucleotide mutations at two separate targeted sites.
• Researchers in Columbia have demonstrated proof-of-concept for using the CRISPR-Cas9 nickase strategy to develop a novel therapeutic alternative for the lysosomal storage disorder mucopolysaccharidosis IVA (MPS IVA). The authors achieved this by knock-in an expression cassette containing GALNS - the gene mutated in the disease - in an in vitro model of MPS IVA. The results showed the successful homologous recombination of the expression cassette into the AAVS1 locus and a long-term increase in GALNS activity, reaching up to 40% of wild-type levels.
• American researchers have used base editing to create CD7-targeting chimeric antigen receptor (CAR T) T cells with four simultaneous point mutations designed for allogeneic use. The authors used cytosine base editors and found that they, in contrast to CRISPR-Cas9, did not impact T-cell proliferation or lead to other abnormalities. In addition, the CAR T cells were shown to be highly efficient against T-cell acute lymphoblastic leukaemia (T-ALL) using multiple in vitro and in vivo models.
• Researchers in China demonstrate how CRISPR-Cas12a-assisted cascade exponential amplification can be used to probe low abundant DNA methylation. The method reveals high sensitivity, which can distinguish as low as 0.01% methylation levels, paving the way towards accelerating methylation-based cancer diagnostics and management.

Industry

• Locus Biosciences announced the completion of a $35 million financing which included several notable participating investors. The proceeds will further develop the company's precision CRISPR-enhanced bacteriophage (crPhage) products to fight deadly infections, including the lead candidate LBP-EC01 for treating urinary tract infections.
• The €480 billion cosmetics industry is looking for greener ingredients, and CRISPR might be part of the solution. In a write-up on Labiotech, Jon Kratochvil at ERS Genomics suggests that the gene-editing technology can be used for the large-scale production of plant-based ingredients like squalane that comes from sharks.
• Fargo-based DNA and mRNA producer Aldevron has signed an agreement to license the manufacturing of Eureca-V Nuclease, a wild-type MAD7 CRISPR Type-V nuclease, from the life sciences company Inscripta. The partnership will aim to offer this nuclease as a standard research-grade and GMP catalogue item.

News from ASGCT

• Several companies have presented new results from ongoing gene-editing studies at the American Society of Gene & Cell Therapy (ASGCT) 25th Annual Meeting that was held 16-19 May in Washington, DC: Precision BioSciences, Poseida Therapeutics, Allogene, Cellectis, Excision BioTherapeutics,Caribou Biosciences, Editas Medicine, Graphite Bio, Metagenomi, Emendo Biotherapeutics and Locanabio.

Detection

• Researchers in China have used a CRISPR-Cas13a signal amplification system to increase the detection sensitivity of specific miRNAs in human serum. The system employs a magnetic relaxation switching (MRS)-based strategy for directly detecting miRNAs in complex samples with a detection limit of 0.22 pM within 60 minutes.
• Chinese researchers have developed a dual-response fluorescent probe that can act as a reporter with CRISPR-Cas12a-based biosensing platforms. The ssDNA probe is dually labelled with fluorescein (FAM) and tetramethylrhodamine (TAMRA). Upon cleavage by activated Cas12a, there will be a decrease in the TAMRA's fluorescence and an increase in the FAM's fluorescence. Compared with the classic TaqMan, the new system exhibited higher sensitivity and could distinguish and avoid false-positive signals.
• Researchers in China have developed a recombinase polymerase amplification-Cas12a-based method for the rapid, in-the-field identification of Bacillus anthracis based on eight novel single-nucleotide polymorphisms (SNPs). B. anthracis is the etiologic agent of anthrax, a fatal disease and a potential biothreat.

Reviews

• A review by Italian researchers discusses the recent progress of electrochemical and optical DNA sensors as promising tools for food safety. Some of the mentioned approaches use CRISPR-Cas9 or -Cas12a to detect pathogens or toxins.
• A review by Jennifer Doudna and colleagues examines the recent advances in understanding the molecular structures and mechanisms of enzymes that comprise bacterial RNA-guided CRISPR-Cas immune systems. The review also discusses how the evolution and structural diversity of CRISPR–Cas systems explain their functional complexity and utility as genome editing tools.
• A book chapter by American researchers describes how to leverage cytosine (BE4max) and adenine (ABEmax) base editors to introduce precise mutations in iPSCs without inducing DNA double-stranded breaks. The chapter also illustrates how to design and clone gRNAs, evaluate editing efficiency, and detect genomic edits at specific sites in iPSCs through base editing technology.
• A review by scientists from India seeks to decrypt the mechanistic basis of the CRISPR-Cas9 protein. The authors discuss how a better understanding of the structural and sequence-based understanding of the whole CRISPR-associated bacterial ortholog family landscape and the underlying energetics of the CRISPR-Cas9 system can reveal critical parameters to design better CRISPR-Cas9s.

Regulation and opinion

• An article in the CRISPR Journal discusses the concept of vulnerability and the ethics concerning human germline genome editing. The authors from Singapore and Australia highlight the role that vulnerability - e.g., situations in which research participants and other individuals may be at a heightened risk of experiencing harm - can play in ethical debates about human heritable genome editing.

News from CRISPR Medicine News

• Monday's feature was about the successful use of CRISPR-Cas9 to selectively disrupt a mutant PSEN1 allele that causes familial Alzheimer’s disease. We interviewed Evangelos Konstantinidis, the first author of the proof-of-concept study, showing that gene editing in human fibroblasts may reverse Alzheimer's disease phenotypes by reducing the accumulation of extracellular amyloid-β deposits.
• On Wednesday, the clinical update reported on a new base-editing cholesterol-reducing therapy that has been approved for a clinical trial in New Zealand. The treatment, VERVE-101, is developed by Verve Therapeutics.
• Our sister-site CRISPR AgroBio News (CARBON) launched its bi-weekly newsletter on Tuesday with links to recent news from CRISPR in agriculture. The top story was the introduction of a single base mutation in SOC1 in lettuce that delayed bolting, i.e., when crops put on a vertical growth spurt to flower and set seed before the vegetables are ready for harvest.