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[Cutting Edge] CRISPR Updates: CRISPR/Cas9 Gene Knockout Technology Helps Develop New Methods to Eradicate Cancer Cells

I.Research News

(Ⅰ)CRISPR Gene Knockout Cells

Researchers at the Shanghai Institute of Silicate, Chinese Academy of Sciences, have recently developed a magnetothermal-induced CRISPR-Cas9 gene editing system to target the knockout of HSP70 and BCL2 genes. This novel approach aims to ablate cancer cells while sparing adjacent healthy cells.

The magnetothermal nanoparticle platform enables plasmid DNA loading, and under the influence of a controlled alternating magnetic field, the mild magnetothermal effect (42°C) not only triggers dual genome editing to disrupt the tumor cells' apoptosis resistance mechanisms but also makes the tumor cells more sensitive to apoptosis through the thermal effect itself, achieving a synergistic therapeutic effect.

This research was recently published in the Journal of Nanobiotechnology under the title "Magnetothermal-activated gene editing strategy for enhanced tumor cell apoptosis."

Original Article Link：https://doi.org/10.1186/s12951-024-02734-8

(Ⅱ)CRISPR Library Screening

Loss-of-function mutations in RNA-binding motif protein 10 (RBM10) are common in lung adenocarcinoma (LUAD) and are often associated with increased tumorigenesis, limiting the efficacy of current LUAD targeted therapies. However, therapeutic strategies utilizing RBM10 deficiency remain unexplored.

To investigate the potential of RBM10 as a treatment target for LUAD, a team from Israel conducted a CRISPR-Cas9 synthetic lethality screen and identified approximately 60 RBM10 synthetic lethality (SL) genes, including the WEE1 kinase. They found that WEE1 inhibition sensitized RBM10-deficient LUAD cells both in vitro and in vivo. Further characterization revealed that RBM10 plays a role in fine-tuning DNA replication.

The study, published in Nature Communications , suggests the potential for targeting tumors with RBM10 deficiencies.

Original Article Link：https://doi.org/10.1038/s41467-024-50882-0

(Ⅲ)Point Mutation

Recognizing the limitations of deaminase-derived DNA base editors in directly editing thymine and guanine, researchers at Peking University have reported utilizing an engineered uracil-DNA glycosylase (UNG) to edit cytosine and thymine bases through a translation DNA synthesis pathway.

On July 30th, in a paper published in Nature Communications , they described how an engineered Deinococcus radiodurans UNG mutant (referred to as DrUNG), when fused with the nickase enzyme Cas9, facilitates efficient editing of endogenous thymine bases, achieving up to 55% editing efficiency without enrichment and exhibiting minimal cytotoxicity.

The team also demonstrated that this novel base editor could restore IDUA enzyme activity in cells derived from patients with Hurler syndrome.

Original Article Link：https://doi.org/10.1038/s41467-024-50012-w

(Ⅳ)CRISPR Detection

1.To repurpose commercial alpha-fetoprotein (AFP) strips for ultrasensitive and handheld nucleic acid detection for diagnostic purposes, researchers at Harbin Institute of Technology have proposed a nucleic acid detection strategy that combines an Exonuclease III/CRISPR-C as12a (Exo-III/Cas12a) cascade amplification strategy with a smartphone-based portable fluorescence detector (SPFD).

The platform demonstrated low detection limits for miRNA-155, Staphylococcus aureus 16S rRNA, and ORF1a/b RNA of the Covid-19 pseudovirus.

The research findings were recently published in Analytical Chemistry.

Original Article Link：https://doi.org/10.1021/acs.analchem.4c02366

2.A research team from Singapore has reported an integrated one-pot proximity isothermal immunoassay incorporating CRISPR-Cas12a for homogeneous protein target detection.

In their setup, probes consisting of different binders conjugated with oligonucleotides undergo bidirectional extension upon binding to the protein target, leading to downstream DNA amplification by a pair of nickases and polymerases, thus generating target sequences for Cas12a signal production.

They demonstrated the feasibility of having all three protein detection elements (target protein binding, DNA amplification, and Cas12a signal generation) coexist in one pot and function isothermally within a single buffer system at a low reaction volume of 10 μL. Their workflow was validated using four different protein targets, with detection limits ranging from fM to pM.

The findings were published in ACS Sensors .

Original Article Link：https://doi.org/10.1021/acssensors.4c00370

(Ⅴ)Others

1.Glaucoma is an incurable disease affecting approximately 80 million people worldwide. Elevated intraocular pressure, the primary risk factor for glaucoma, is determined by the balance between the secretion and outflow of aqueous humor.

Recently, scientists from the UK and China published an article in Nature Communications demonstrating that the RNA interference tool CasRx can reduce the expression of aqueous humor circulation-related genes Rock1 and Rock2, as well as aquaporin 1 and β2-adrenergic receptor in female mice.

This strategy significantly lowered intraocular pressure in female mice, provided protection to retinal ganglion cells, and slowed disease progression.

Original Article Link：https://doi.org/10.1038/s41467-024-50050-4

2.Israeli scientists have developed a deep learning model called DeepCRISTL, which can predict editing efficiency in specific cellular environments.

The new tool uses high-throughput datasets to learn general patterns of gRNA editing efficiency, then fine-tunes the model based on functional or endogenous data to adapt to specific cellular contexts.

The team tested two state-of-the-art models (DeepHF and CRISPRon) trained on high-throughput datasets, using various transfer learning methods for editing efficiency prediction. They found that the combination of CRISPRon and fine-tuning all model weights performed the best overall.

Their findings were published earlier this week in Bioinformatics .

Original Article Link：https://doi.org/10.1038/s41467-024-50050-4

II.Industry News

1.Intellia Therapeutics recently announced that the UK Medicines and Healthcare products Regulatory Agency has approved the clinical trial application for NTLA-3001. NTLA-3001 is a CRISPR-based in vivo therapeutic candidate currently under development for the treatment of alpha-1 antitrypsin deficiency.

2.Cellectis announced on August 1st that the FDA has granted orphan drug designation to CLLS52 (alemtuzumab). CLLS52 is an investigational product used as part of a lymphocyte depletion regimen associated with Cellectis' therapeutic candidate UCART22, and was evaluated in the BALLI-01 clinical trial for relapsed/refractory B-cell acute lymphoblastic leukemia.

3.AIRNA, a pioneering biotech company providing RNA editing therapies for rare and common diseases, recently announced the completion of an oversubscribed $60 million funding round, bringing the total amount raised in its Series A round to $90 million. According to the press release, the funds led by Forbion will be used to advance AIRNA's primary candidate for alpha-1 antitrypsin deficiency into clinical trials and further develop AIRNA's extensive product pipeline.

4.Precision Biosciences reported its Q2 2024 financial performance and business updates in a press release on August 1st. Among the updates, the company announced that its wholly-owned projects PBGENE-HBV for chronic hepatitis B virus and PBGENE-PMM for m.3243 mitochondrial disease are expected to have clinical trial applications submitted in the US and EU in 2024 and 2025, respectively.

5.Vertex Pharmaceuticals released its Q2 2024 financial results on August 1st. The company disclosed that it has sufficient funds to achieve Phase 1 clinical data for multiple in vivo gene editing projects, with cash flow expected to last into the second half of 2026. The company also reported regaining control over three advanced preclinical projects for internal or partnered development, including a novel gene editing approach for Duchenne muscular dystrophy.

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