CMN Weekly (17 May 2024) - Your Weekly CRISPR Medicine News

Top picks

• Greek and German researchers haven developed BreakTag, a versatile method for profiling Cas9-induced DNA double-strand breaks (DSBs) and identifying the determinants of Cas9 incisions. They found that approximately 35% of SpCas9 DSBs are staggered and that the type of incision is influenced by DNA:gRNA complementarity and the use of engineered Cas9 variants.
• CRISPR-Cas9 has been used to knock out and overexpress MICU3 - a regulatory protein involved in mitochondrial calcium uptake - in mice. Knockout hearts showed reduced mitochondrial calcium uptake ([Ca2+]m) after adrenergic stimulation, while overexpression increased [Ca2+]m but led to decreased cardiac function and hypertrophy. MICU3 levels were lower in failing human hearts, indicating its crucial role in cardiac [Ca2+]m regulation and potential as a therapeutic target.

Research

• CRISPR-Cas9 knockdown of Aquaporin-8 (AQP8) - involved in glioma proliferation and tumour growth - has been demonstrated to inhibit H2O2 release, increase mitochondrial ROS, and impair mitochondrial function. This led to reduced cell proliferation, G0/G1 phase arrest, and elevated p53/p21 expression, highlighting AQP8 as a potential target for glioma treatment.
• An American study has used prime editing to target mutated leptin receptors in B6.BKS(D)-Leprdb/J mice (db/db mice) by injection into the flexor digitorum brevis muscle. The treated skeletal muscle exhibited enhanced leptin receptor signals, demonstrating in vivo efficacy and potential for functional protein rescue.
• A Japanese study fused H2A.X - a histone variant involved in DNA repair processes - to Cas9, improving the HDR/NHEJ ratio by suppressing NHEJ. While H2A.X mimicry variants were ineffective, H2A.1 showed superior NHEJ suppression, highlighting histone-Cas9 fusions as a promising precision editing strategy.
• Thai researchers have developed a combined gene-editing approach using CRISPR-Cas9 and C46, a cell membrane-anchored HIV-1 fusion inhibitor, to enhance cellular HIV resistance. This dual method offers a promising avenue for treating HIV-1 by integrating multiple resistance mechanisms within a single treatment strategy.
• Scientists in South Korea have demonstrated enhanced editing efficiency in hPSCs by dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by cytosine base editor or prime editor.
• A recent study provides significant insights into how specific long noncoding RNAs (lncRNAs) influence breast cancer risk by affecting cell proliferation. Utilising CRISPR-Cas13d technology, researchers have identified and characterised the impact of the lncRNA KILR, which plays a role in controlling DNA replication and repair processes.
• Italian researchers have examined the role of the Wnt signalling modulator Dickkopf-1 (DKK1) in metastatic castration-resistant prostate cancer (mCRPC) using siRNA and CRISPR-Cas9. Silencing or deleting DKK1 reduced cell growth, migration, and metastasis-associated gene expression in PC3 and DU145 cells. Moreover, it decreased tumour growth and lung metastases in xenograft models.

CRISPR screens

• A CRISPR screen has identified PPP6C as crucial for MAPK inhibitor sensitivity in KRAS- and BRAF-mutant colorectal cancer and melanoma cells. PPP6C deletion increased resistance to MAPK inhibitors by enhancing NF-κB signalling. Inhibiting NF-κB restored drug sensitivity. A PPP6C R264 mutation had similar effects. Targeting the PP6-NF-κB axis may improve treatment for PPP6C-mutant cancers.
• Using CRISPR screening, Chinese scientists have identified PLAG1 as a critical gene conferring resistance to ferroptosis in hepatocellular carcinoma (HCC) cells treated with sorafenib. Sorafenib induces ferroptosis, an iron-dependent cell death, by regulating PLAG1 through the lncRNA PVT1, affecting GPX4 expression and redox balance. Targeting PLAG1 could enhance sorafenib's effectiveness in HCC therapy.
• Genome-wide CRISPR screens in spheroid culture reveal that the tumour suppressor LKB1 inhibits growth via the PIKFYVE lipid kinase. The American scientists further used chemical inhibitors and a pH-sensitive reporter to determine that LKB1 impairs growth by promoting the internalisation of wild-type EGFR in a PIKFYVE-dependent manner.

Industry

• Sangamo Therapeutics announces first quarter 2024 financial results with a net loss of $49 million and $54 million in cash.
• Prime Medicine announces first quarter 2024 financial results with a net loss of $46 million and $224 million in cash.
• Poseida Therapeutics announces first quarter 2024 financial results with a net loss of $24 million and $199 million in cash.
• Beam Therapeutics announces first quarter 2024 financial results with a net loss of $99 million and $1,100 million in cash.

Detection

• Chinese researchers have developed a one-pot CRISPR-Cas12a-RCA strategy for ultrasensitive and specific detection of circRNA. The method utilises reverse transcription-rolling circle amplification (RT-RCA) and can detect circRNAs at a concentration as low as 300 aM in 30 minutes to 2 hours.

Reviews

• CRISPR/Cas-based colorimetric biosensors: A promising tool for diagnosis of bacterial foodborne pathogens in food products. This review classifies and discusses the working principles of the different CRISPR-Cas protein agents that have been employed in biosensors so far and assesses the current status of the CRISPR system, specifically focusing on colourimetric biosensing platforms.
• CRISPR-Cas9 delivery strategies and applications: Review and update. This review reviews the CRISPR system, focusing on the history, classification, delivery methods, applications, new generations, and challenges of CRISPR-Cas9 technology.
• CRISPR-powered RNA sensing in vivo. This review reclassifies RNA sensors into four categories based on their working mechanisms and discusses the advantages and challenges of existing methodologies in diverse application scenarios.
• Advancements in CRISPR-Based Therapies for Genetic Modulation in Neurodegenerative Disorders. The review delves into the foundational principles of CRISPR technology, highlighting its unparalleled ability to edit genetic sequences with unprecedented precision.

Conferences

• The patient-led charity Fighting Blindness will host the Retina International World Congress (RIWC24) in Dublin from June 5-8, 2024. The congress will bring together the world's foremost retinal scientists and clinicians and will include two presentations about gene-editing approaches to treat retinal disorders.
• The State of CRISPR and Gene Editing 2024 is a virtual summit that will be broadcast on Wednesday, June 5, 2024, at 8:00 a.m. PT. Highlights include an opening keynote from David Liu and talks and fireside chats about the latest research developments, innovations, and advanced technologies that are expanding the CRISPR toolbox, delivering new therapies to patients, and safeguarding our food supply. Registration is free.
• Save the date, April 7-11th, 2025, for CRISPRMED25 in Copenhagen. CRISPR Medicine News will host the event where you can meet the global CRISPR Medicine community and dive into presentations and discussions about Tools, Delivery, Safety/Off-target, Diseases, Pre-clinical/clinical, Standards and Regulations, Functional Genomics, Target Identification and Target Validation.
• You can now register for the 2nd CRISPR-Based Therapy Analytical Development Summit in Boston September 17-19, 2024. The presentations will focus on: Characterisation of guides, nucleases, and full drug constructs; Robust non-clinical and CMC assay development, including biodistribution studies, potency assays, and on-/off- target sequencing detection, nomination, and prediction; Meeting regulatory expectations for analytical data to successfully support IND submissions

News from CRISPR Medicine News

• In a clinical update Wednesday, we shared clinical data presented at the ASGCT meeting in Baltimore last week, revealing that Excision Biotherapeutics' CRISPR-based therapy for HIV, EBT-101, is safe but doesn't cure HIV. EBT-101 met the primary and secondary endpoints of safety and biodistribution/ immunogenicity, respectively. Still, it did not prevent viral rebound in three individuals who stopped anti-retroviral medication in a Phase 1/2 clinical trial.