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

Top picks

• American researchers have introduced Precise RNA-mediated Insertion of Transgenes (PRINT), a novel method for inserting transgenes directly into the genome at a specific safe-harbour locus. PRINT leverages two RNAs: one encodes the avian R2 retroelement protein for target site recognition and nicking, and another encodes the transgene. This approach, tested in human primary cell lines, achieved over 50% efficiency in inserting multiple 2kb transgenes with minimal risk of off-target mutations and immune responses, highlighting its potential for safe and scalable genetic modifications.
• The new MEGA platform, leveraging CRISPR-Cas13d, advances T-cell therapies by offering a safe, efficient, and broad-spectrum method for editing the T-cell transcriptome. RNA-guided RNA degradation allows for reversible and highly multiplexed gene knockdown in human T cells without DNA alterations. Applied to CAR T cell exhaustion, MEGA effectively mitigates inhibitory receptor expression and identifies key T cell regulators, enhancing CAR T cell efficacy against tumours both in vitro and in vivo.

Research

• A new study finds that CRISPRa-activated genes in human pluripotent stem cells are silenced in differentiated cells. The study found that initially successfully activating genes in pluripotent cells, the system faced an unexpected challenge: the expression of the dCas9-activator complex was silenced when cells differentiated into cardiomyocytes and endothelial cells.
• Natural mechanisms of cancer resistance in mammals may have been elucidated in a study that used CRISPR-Cas9 to identify genes that render bats exceptionally resistant to cancer. The authors used the gene-editing technology to inhibit the expression of HIF1A, COPS5, and RPS3 and found that their suppression significantly inhibited cell proliferation. The results suggest that the downregulation of these genes may be involved in the resistance of bat cells to malignant transformation.
• A Chinese study presents a novel method for detailed analysis of on-target CRISPR-Cas9 gene editing, using nanopore sequencing to characterise complex DNA insertions. The technique applied to haemophilia A gene therapy in mice revealed expected F8 gene insertions and unexpected large and diverse fragment insertions, including genomic DNA and LINE-1 elements.
• Researchers in China describe a universal gene-editing approach to conquer the diverse gain-of-function mutations causing autosomal dominant retinitis pigmentosa (adRP). They have developed a CRISPR-Cas12i-based, mutation-independent gene knockout and replacement compound therapy carried by a dual AAV8 system that successfully delayed the progression of retinal degeneration in the classic mouse disease model RhoP23H.
• A novel method for enhancing mRNA translation without changing transcription levels has been achieved by fusing the catalytically inactive Cas13d with the eIF4G translation regulatory element. The new CRISPR-dCas13d-eIF4G system was applied to a kidney stone disease model with specifically increased GPX4 protein expression in renal cells, and it effectively countered ferroptosis and reduced calcium oxalate crystal-induced damage.
• A Chinese study showcases a CRISPR-Cas9-based system to counteract resistance to "last resort" antibiotics tigecycline and colistin in Gram-negative bacteria. By targeting resistance genes tet(X4) and mcr-1 with high specificity, the system achieved over 90% resensitisation of clinical isolates and reduced resistant bacteria counts to 1% in vivo.

Industry

• A news feature in Biopharma Dealmakers describes how CRISPR partnerships propel precision medicine. In a significant leap for precision medicine, major biopharma giants are joining forces with CRISPR technology frontrunners to harness the revolutionary potential of genome editing.
• Intellia Therapeutics provides 2023 financial results with a net loss of $132.2 million for the fourth quarter and $1.0 million in cash at the end of the year.
• CRISPR Therapeutics provides 2023 financial results with a net income of $89.3 million for the fourth quarter and $1,696 million in cash at the end of the year.
• ProQR Therapeutics has successfully defended against an opposition filed in Japan against a patent directed to its ADAR-mediated RNA editing platform Axiomer. The Japanese Patent Office rejected the opposition and indicated that all claims were to be maintained as granted to ProQR.
• Iovance Biotherapeutics announced the pricing of an underwritten offering of 23,014,000 shares of its common stock at an offering price of $9.15 per share. The gross proceeds from the offering are expected to be approximately $211 million.
• Precision BioSciences announces a non-exclusive patent license agreement that grants Caribou Biosciences a non-exclusive, worldwide license to one of Precision's foundational cell therapy patent families for use with CRISPR in human therapeutics. The licensed patents relate to the targeted insertion of a sequence encoding an exogenous antigen binding receptor into the T cell receptor alpha constant (TRAC) gene locus of human T cells via a single gene edit.
• Prime Medicine announced the closing of its underwritten upsized public offering of 22,560,001 shares of its common stock for $6.25 per share. The gross proceeds to Prime Medicine were approximately $161.0 million.

Detection

• Researchers in Thailand have introduced the Ov-RPA-CRISPR-Cas12a assay for detecting Opisthorchis viverrini - a parasite in the bile duct - infection in human faeces. This novel assay, combining recombinase polymerase amplification with CRISPR-Cas12a, demonstrated the ability to detect O. viverrini DNA at very low concentrations, providing a promising tool for field diagnostics and enhancing the accuracy of infection identification, crucial for effective treatment and control measures.
• A new sensitive heparin detection method leverages the finding that heparin, a common anticoagulant, can inhibit the Cas12 protein in CRISPR systems by competing with crRNA for Cas12 binding. This interaction, driven by heparin's size and charge, has been harnessed to develop the new detection system with a fluorometric limit of 0.36 ng/mL and a rapid, 20-minute lateral flow test, HepaStrip.

CRISPR screens

• Chinese researchers describe a programmable method for large-scale functional circRNA screening based on the RNA-guided, RNA-targeting CRISPR-Cas13 (RfxCas13d) system. The technique can be applied both in vivo and in cells to explore highly expressed circRNAs that may influence cell growth under natural conditions or in response to environmental stimulation without disturbing cognate linear mRNAs.

Reviews

• CRISPR/Cas9: an overview of recent developments and applications in cancer research. This review article provides an extensive overview of the research that has been done so far on CRISPR-Cas9 with an emphasis on how it could be utilised in the treatment of cancer.
• Recent advances in CRISPR-Cas9-based genome insertion technologies. This review summarises recent advances in programmable genome insertion techniques and elaborates on the cons and pros of each method. Moreover, it identifies opportunities for future improvements and applications in basic research and therapeutics.
• On the ever-growing functional versatility of the CRISPR-Cas13 system. This review discusses the functional versatility of the CRISPR-Cas13 system, which includes the ability for RNA silencing, RNA editing, RNA tracking, nucleic acid detection, and translation regulation.
• Non-viral delivery of nucleic acid for treatment of rare diseases of the muscle. This review focuses on non-viral vectors for delivering therapeutic cargoes to treat muscular dystrophies such as Duchenne muscular dystrophy (DMD), GNE myopathy, spinal muscular atrophy (SMA), and limb-girdle muscular dystrophy.
• Rare genetic disorders in India: Current status, challenges, and CRISPR-based therapy. This review explores the landscape of rare genetic diseases in India, along with national policies and significant challenges. It examines the implications of CRISPR-based therapies for providing personalised and effective treatments.

Perspectives

• A news feature in Nature, "Move over, CRISPR: RNA-editing therapies pick up steam", describes how two RNA-editing therapies for genetic diseases have gained approval for clinical trials in the past few months, raising hopes for safer treatments. The RNA-editing approaches differ in the two trials: One is exploring single-base editing to treat a genetic lung and liver disorder called alpha-1 antitrypsin deficiency (AATD), while the other approach, called RNA exon editing, changes thousands of genetic letters in an RNA molecule at once and aims at treating disorders caused by multiple mutations in a person's genome.

News from CRISPR Medicine News

• On Monday, we interviewed two Chinese scientists behind a recent breakthrough in gene-editing technology that introduces circular RNA-mediated prime editors (CPEs), harnessing the Cas12a system to offer an innovative alternative to the conventional CRISPR-Cas9 approach. With four distinct CPE variants demonstrating up to 40% editing efficiency in human cells without selection, this development marks a significant stride forward in the precision and versatility of genome editing, promising new avenues in biological research and therapeutic interventions.