CMN Weekly (23 August 2024) - Your Weekly CRISPR Medicine News

Top picks

• CRISPR-Cas editing near the sickle cell mutation in the human globin gene locus triggers unintended genetic rearrangements, new research suggests. These rearrangements, marked by a δ-globin gene footprint near the cut site, occur without an external DNA template and result from double-stranded breaks. The findings highlight the risks of DNA repair mechanisms during gene editing, particularly in sequence-related gene families.
• Japanese researchers show that CRISPR-Cas9 editing significantly increases genome-wide mutations, particularly C to A substitutions, in resting TSCE5 cells, inducing 859 mutations compared to 16 in unedited cells. Under S∼G2/M arrest and cyclin-dependent kinase 1 inhibition, only five mutations occurred. Transcriptomic analysis revealed altered DNA repair pathways, highlighting the impact of CRISPR on genome-wide mutation processes and the activation of alternative repair mechanisms to maintain genome integrity.

Research

• A Danish study details the development of virus-derived particles to deliver CRISPR-Cas9 ribonucleoprotein complexes for targeted gene editing. These particles enable in vivo editing in specific tissues, including the retina, liver, and brain, as well as primary cells like T-cells and haematopoietic stem cells. The particles successfully carry base and prime editors, highlighting their therapeutic potential for large-scale clinical applications.
• A new CRISPR-dCas13 tool developed by French researchers modulates alternative splicing without altering gene expression. The RNA-editing system, based on the 'dead' Cas13 enzyme, targets splicing regulators in endogenous transcripts to switch between splicing isoforms.
• American researchers have created a computational model to simulate CRISPR-Cas9-mediated barcode editing during lineage tracing, specifically tracking the effects of mutation accumulation over generations. The simulation focused on how barcode mutation profiles emerge, their impact on reconstructing accurate lineage trees, and factors influencing lineage accuracy. Key findings show that sampling size, indel probabilities, and large segment deletions in early generations are critical to improving the precision of lineage tracing methodologies, particularly for complex organisms.
• A Chinese study shows that fusing multivalent molecules like intrinsically disordered regions (IDRs) and modular domains (MDs) to the dCas9-VP64 activator enhances transcriptional activation. Optimal combinations of these molecules and guide RNA sites increase efficiency while targeting enhancer-promoter pairs further boosts activation through chromatin interactions. This platform improves gene regulation, offering potential for therapeutic applications.
• CRISPR-Cas9 nicking enzymes have been used to explore the impact of replication fork collisions with DNA nicks. By inducing specific nicks, it demonstrated that leading-strand fork collapse generates single-ended double-strand breaks (seDSBs), while lagging-strand nicks can result in double-ended DSBs (deDSBs). The research highlights how distinct repair mechanisms are triggered based on the strand affected during replication.
• Chinese researchers have used CRISPR-Cas9 for gene knockout and overexpression to show that CXXC5 silencing reduces proliferation and promotes differentiation, while overexpression has the opposite effect. Proteomic analysis further revealed CXXC5's influence on key processes like apoptosis. In vivo, CXXC5 silencing impairs haematopoietic stem cell (HSC) engraftment, highlighting its therapeutic potential for haematological diseases.
• Using CRISPR-Cas9 genome screening, a new study identifies a novel mechanism linking perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) exposure to non-alcoholic fatty liver disease (NAFLD). The study reveals that PFOA and PFOS at human-relevant doses downregulate nudt7 transcription, reducing Ace-CoA hydrolase activity and promoting lipid accumulation in HepG2 cells. This mechanism potentially explains the increased NAFLD risk associated with polyfluoroalkyl substance (PFAS) exposure in humans.
• South Korean researchers demonstrate that combining RAD51, a key factor in homologous recombination, with SCR7 significantly enhances CRISPR-Cas9 genome editing efficiency by promoting homologous recombination-based DNA repair. RAD51 improves knock-in/knockout efficiency and insertion/deletion mutations, while SCR7 aids in resolving R-loops. This approach boosts CRISPR-Cas9 accuracy and stability, offering promising applications in biotechnology and therapeutics.

Industry

• Following a $213 million fundraising in 2023, Tome Biosciences is scaling back operations as investor sentiment in the gene editing sector, especially for preclinical companies, has shifted. The company is operating at reduced capacity, exploring strategic options, and possibly seeking a buyer, though details on employee impacts or changes to its pipeline remain undisclosed.
• Aldevron has shared a new whitepaper, Next-Generation CRISPR Approaches, describing how recent FDA and EMA approvals for CRISPR-based therapies have ignited a wave of innovation in cell and gene therapy development. Navigating these advancements requires a deep understanding of the diverse manufacturing approaches and their unique requirements.

CRISPR screens

• Italian researchers have used CRISPR-Cas9 screening to identify long non-coding RNA (lncRNA) RP11-350G8.5 as a key oncogenic factor in multiple myeloma (MM). The team employed CRISPR-Cas9 technology to conduct an extensive and unbiased loss-of-function screen, systematically disrupting 671 lncRNAs in both drug-sensitive and bortezomib-resistant MM cell lines. The study suggests RP11-350G8.5 as a novel therapeutic target, particularly in drug-resistant cases.
• American researchers have used CRISPR screening to identify immunometabolic regulators in CD4 T cells by targeting genes involved in inborn errors of metabolism (IEMs) and immunity (IEIs). The screen uncovered significant overlap between IEM and IEI genes, highlighting metabolic vulnerabilities in T cells, such as the role of Gfpt1 in UDP-GlcNAc synthesis for T cell function.
• A new study identifies a resistance mechanism to Menin inhibitors in KMT2A-rearranged leukaemia through chromatin-focused CRISPR screens. The screen revealed that the loss of PRC1.1 subunits reactivates non-canonical Menin targets like MYC. Targeting MYC or combining Menin inhibitors with BCL2 inhibitors (venetoclax) resensitises resistant leukaemia cells, offering potential strategies to treat PRC1.1-deficient AML.
• A CRISPR interference screen has identified enhancers relevant to diabetes during pancreatic differentiation in human pluripotent stem cells (hPSCs). The ONECUT1e-664kb enhancer was found to regulate ONECUT1 expression and its deletion impaired pancreatic differentiation. A type 2 diabetes-associated variant (rs528350911) in ONECUT1e-664kb disrupts the binding of key transcription factors, highlighting its causal role in diabetes and the importance of enhancer discovery for disease research.

Detection

• Chinese researchers have developed CLEAR, a CRISPR/Cas13a-Cas12a-based lateral flow assay for ultrasensitive, rapid at-home detection of SARS-CoV-2 RNA without nucleic acid amplification. CLEAR achieves 1 aM sensitivity and 100% accuracy in clinical swabs. It provides a low-cost, user-friendly solution that addresses the limitations of traditional PCR systems and enables efficient variant detection for widespread public health use.

Reviews

• Advances in manufacturing chimeric antigen receptor immune cell therapies. This review focuses on advancements in CAR immune cell manufacturing, highlighting innovations in automation, process development, and the application of CRISPR gene editing to improve scalability and efficiency in cell therapy production.
• Next-generation CRISPR technology for genome, epigenome and mitochondrial editing. This review focuses on the current CRISPR toolboxes, examining their potential and challenges in nuclear, epigenome, and mitochondrial editing, as well as ethical and societal considerations in CRISPR-based gene therapies.
• Emerging DNA & RNA editing strategies for the treatment of epidermolysis bullosa. This review discusses the status quo of current gene therapeutics that act at the level of RNA or DNA, all with the common aim of improving the quality of life for EB patients.

Huh, Heh, Wow

• The Guardian reports that the world's first mRNA lung cancer vaccine, BNT116 from BioNTech, has entered clinical trials across seven countries. Designed to treat non-small cell lung cancer, the vaccine primes the immune system to recognise tumour markers and attack cancer cells. Around 130 patients, ranging from early-stage cases prior to surgery or radiotherapy to those with advanced or recurrent cancer, will receive the vaccine in combination with immunotherapy. Patients receive six RNA-based injections - each containing different RNA strands - over 30 minutes, followed by weekly vaccinations for six weeks, and will then continue with doses every three weeks for a total of 54 weeks.

News from CRISPR Medicine News

• On Monday, we brought a mini-interview with two Israeli researchers who have contributed to the development of CRISPECTOR2.0, an enhanced version of the CRISPECTOR tool. CRISPECTOR2.0 offers improved accuracy by integrating a reference-free method for detecting allele-specific activity and quantifying off- and on-target effects. Thereby, it addresses the challenge of off-target effects in genome editing, which vary among individuals due to genetic diversity. We previously wrote about CRISPECTOR here.
• On Wednesday, we reported new favourable pharmacokinetic and safety data from US-based Locus Biosciences concerning the company's ongoing Phase 2 trial of CRISPR-edited phage therapy candidate LBP-EC01. LBP-EC01 is being developed as a novel treatment for uncomplicated urinary tract infections caused by antimicrobial-resistant and multi-drug-resistant E. coli. The data from the randomised, uncontrolled, open-label trial were published in The Lancet Infectious Diseases.