CMN Weekly (22 September 2023) - Your Weekly CRISPR Medicine News

Top picks

• On Monday, we officially launched The First CRISPR Medicine Conference!! Registration and abstract submission is now open at our dedicated CRISPRMed2024 website, with early-bird pricing until 1st December 2023. Join us in Copenhagen in April 2024 and meet the CRISPR Medicine community from the academic and industry segments, join the sessions, the workshops and the network meetings to get the latest updates, and find your next collaboration at the social events.
• In an article published in Nature this week, researchers led by Randall Platt at ETH Zürich introduced AAV-Perturb-seq, a direct in vivo single-cell CRISPR screening method that can profile transcriptional phenotypes across perturbations and cell types. AAV-Perturb-seq is expected to simplify and accelerate research with laboratory animals. The method exploits CRISPR-Cas to make many gene edits in the cells of a single animal, simultaneously, much like a mosaic. While only a single gene is edited in each cell, the various cells within an organ collectively harbour a range of edits. This allows the analysis of edits in single cells, and makes it possible to study the impact of many different edits in a single experiment.

Research

• In yesterday's edition of PNAS, a global team of researchers report a genetic system for deliberately inducing a decrease in the abundance of a target organism in a gut community. In their study, mice (in which all microorganisms present were known) were fed a human diet and colonised with a defined community of cultured, genome-sequenced, human gut bacteria, which included multiple Bacteroidaceae species. CRISPR-mediated knockdown of different Bacteroides within the established community resulted in specific increases in the abundances of other organisms and revealed how they dynamically changed their priorities for utilising available polysaccharides while preserving the community’s overall capacity to metabolize carbohydrates. The work is important because human gut Bacteroidaceae are primary consumers of complex dietary polysaccharides, and understanding how our gut bacteria compete and cooperate to metabolise nutrients is a crucial step towards developing more nutritious foods and promoting healthier gut microbial communities.
• In an article published in Nature Scientific Reports earlier this month, scientists at University of Iowa (US) compare four widely used INDEL software programmes designed for the analysis of in vivo genomic engineering: TIDE (developed at Netherlands Cancer Institute), ICE Analysis (Synthego), DECODR (Deconvolution of Complex DNA Repair, developed at ChristianaCare Gene Editing Institute), and Indigo (developed at EMBL). By analysing the same genetic target across a wide panel of cell lines with the same sequence file, the researchers could make systematic conclusions about the differences in the four packages for analysis of in vivo-generated indels. They report high variability in the reported number, size, and frequency of indels across each software platform, and highlight the importance of selecting indel analysis platforms specific to the context that the gene-editing approach is being applied.
• A team led by Ben Kleinstiver at Mass General Hospital and Harvard Medical School recently reported click editing, a novel genome-writing platform that combines the beneficial properties of DNA-dependent DNA polymerases with RNA-programmable nickases (e.g. CRISPR-Cas) to permit the installation of a range of genomic edits including substitutions, insertions, and deletions, using a tethered ssDNA template that they call a clkDNA. The team expects that the scalability and versatility of click editing will make it broadly useful to researchers for a wide range of applications. Their findings were shared last week in a manuscript on the pre-print server bioRxiv. In an article published the same week in Nature Biotechnology, researchers led by Erik Sontheimer and Wen Xue (both at University of Massachusetts) independently reported the activity of a DNA-dependent DNA polymerase (phi29), untethered from Cas9, which enables editing from a synthetic, end-stabilised DNA-containing template at up to 60% efficiency in human cells. They find that compared to prime editing, DNA polymerase editing avoids autoinhibitory intramolecular base pairing of the template, facilitates template synthesis and supports larger insertions (>100 nucleotides).
• In an article published yesterday in Nature Chemical Biology, scientists at University of Zurich and ETH Zurich report the use of phage-assisted continuous directed evolution to broaden the PAM compatibility of Campylobacter jejuni Cas9 (CjCas9), which is the smallest Cas9 ortholog characterised to date. They found that the new variant, evoCjCas9, primarily recognises certain PAM sequences that occur tenfold more frequently in the genome than the canonical N₃VRYAC PAM site. They also found that evoCjCas9 exhibits higher nuclease activity than wild-type CjCas9 on canonical PAMs, with editing rates comparable to commonly used PAM-relaxed SpCas9 variants. The newly identified variant was also demonstrated to elicit robust base and prime editing, when combined with deaminases or reverse transcriptases, respectively.
• Researchers based at Rice University (US) report detailed biochemical and structural characterisations of a compact Cas13 (Cas13bt3) suitable for adeno-associated virus (AAV) delivery. In contrast to many other Cas13 systems, Cas13bt3 cleaves the target and other non-specific RNA at internal "UC" sites and is activated in a target length-dependent manner. The cryo-electron microscope structure of Cas13bt3 in a fully active state illustrates the structural basis of Cas13bt3 activation. Guided by the structure, the team generate engineered Cas13bt3 variants with minimal off-target cleavage that maintain target cleavage activities. Their findings were published this week in Nature Communications.
• In PLoS Genetics this week, scientists in China report the crystal structure of Cas12g, an endonuclease that belongs to the type V RNA-guided CRISPR-Cas family. Cas12g is known for its ability to cleave RNA substrates using a conserved endonuclease active site located in the RuvC domain. In their study, the team determined the crystal structure of apo-Cas12g, the cryo-EM structure of the Cas12g-sgRNA binary complex and investigated conformational changes that occur during the transition from the apo state to the Cas12g-sgRNA binary complex. Overall, their work reveals key structural transitions upon gRNA binding and the importance of these for Cas12g-mediated RNA cleavage. Read their full findings here.
• Researchers in Japan present NICER, a method to correct heterozygous mutations that employs multiple nicks (MNs) induced by Cas9 nickase and a homologous chromosome as an endogenous repair template. They find that NICER rarely induces unintended genomic alterations, and that it can restore the expression of disease-causing genes in cells derived from genetic diseases with compound heterozygous mutations. You can read their full findings in their recently-published article in Nature Communications.
• In an article published recently in Nature Communications, a team in the US present a split adenine base editor (sABE) that utilises chemically-induced dimerisation (CID) to control the catalytic activity of the deoxyadenosine deaminase TadA-8e. They demonstrate that ABE is capable of high on-target editing activity comparable to the original ABE with TadA-8e (ABE8e) upon rapamycin induction, while maintaining low background activity without induction. They also find that sABE exhibits a narrower activity window on DNA and higher precision than ABE8e, with an improved single-to-double ratio of adenine editing and reduced genomic and transcriptomic off-target effects.

Industry

• Integra Therapeutics presented ex vivo and in vivo data for its FiCAT gene-writing platform at the recent Advanced Therapies Europe congress held in Portugal. The data demonstrates the potential of FiCAT technology to write large and small genes to design safe, effective cell and gene therapies, and opens the door to the regulatory phase. You can learn more about the FiCAT platform in our interview with Integra CSO and CEO here.
• AIRNA, a biotech company pioneering RNA-editing therapeutics to restore the health of patients with rare and common diseases, emerged from stealth earlier this week with $30 million in initial financing led by ARCH Venture Partners. This funding will allow AIRNA’s team to advance a pipeline of RNA-editing therapeutics driven by its proprietary RNA-editing platform, RESTORE+^TM. AIRNA is a spinout from Tübingen University (Germany) and Stanford University (US) and its first therapeutic programme aims to develop a best-in-class candidate to treat the inherited genetic disease alpha-1 antitrypsin deficiency.
• Cambridge-based start-up Broken String Biosciences announced this week that it has closed a $15 million Series A financing round. Illumina Ventures and Mérieux Equity Partners led the round, joined by Heran Partners, Tencent, and Dieter von Holtzbrinck Ventures. The company plans to use the financing to develop and commercialise its next-gen sequencing-based technology Induce-seq, which it has developed for DNA break mapping. Broken String Biosciences was spun out of Cardiff University (Wales) in 2020.

Reviews

• Drug delivery systems for CRISPR-based genome editors. Gene-editing therapies can in theory edit or correct any genetic sequence, offering hopes of functional cures for rare genetic diseases, cancers, chronic metabolic diseases, and countless others. However, for these therapies to be effective AND safe, they must be able to reach the target cell without provoking an unwanted immune response. This is where delivery systems come into the picture. In a review published earlier this week, researchers at various US institutes provide a timely overview of drug delivery systems for CRISPR-based genome editors, focusing on adeno-associated viruses and lipid nanoparticles. The authors describe how these systems have been engineered and characterised in pre-clinical animal models, and highlight data from recent clinical trials. They also discuss pre-clinical targeting mediated by polymers, proteins, including virus-like particles, and other delivery vehicles that may deliver gene-editing therapeutics in the future.
• CRISPR delivery with extracellular vesicles: Promises and challenges. In this review, researchers in Denmark and Spain provide a comprehensive analysis of the currently available strategies for extracellular vesicle (EV)-mediated delivery of CRISPR-Cas9 gene-editing reagents. These strategies include cell-based, passive loading obtained by overexpression of CRISPR-Cas9, active loading involving protein or RNA dimerisation, and loading into already purified EVs.
• RNA-based medicine: from molecular mechanisms to therapy. In this review, authors in Germany present an overview of different RNA-based strategies to generate novel therapies, including antisense and RNAi-based mechanisms, mRNA-based approaches, and CRISPR-Cas-mediated genome editing. Using three rare genetic diseases as examples, they highlight the opportunities and challenges to wide-ranging applications of RNA therapeutics.

Detection

• Streptococcus suis serotype 2 is an economically important zoonotic pathogen that causes septicemia, arthritis, and meningitis in pigs and humans. In an article published recently in Talanta, scientists in China and the UK report the development of a high-fidelity detection and serotyping platform for S. suis serotype 2 based on recombinase polymerase amplification (RPA) and CRISPR-Cas12a system. The assay, which they call Cards-SSJ/K, was demonstrated to have a detection limit of 10 CFU, takes <60 min, and no cross-reaction was found with other S. suis serotypes, closely related Streptococcus spp., or common pig pathogens, and Cards-SSK could differentiate between very closely related serotypes. The authors propose that despite the increased cost of their approach, compared to current PCR-based methods, its speed and low resource demands makes it an excellent choice for point-of-care detection for S. suis serotype 2.
• In an article published yesterday in Analytical Chemistry, scientists in China present a new isothermal fluorescence amplification device that was built for the rapid detection of pathogens during public health crises. The device features multichannel capability for simultaneous detection of various targets, integrates with the Internet of Medical Things (IoMT) for remote control and data uploading, and includes a deep learning-based batch processing system for rapid (9.4 ms) and accurate discrimination of pathogen type with excellent accuracy. During its development and validation, the team successfully applied the device to simultaneously detect Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) with limits of detection (LODs) of 18 CFU/mL (SA) and 20 CFU/mL (MRSA) within 35 min by multiplex RPA assay and CRISPR/Cas12a-mediated nucleic acid detection assay.
• Recognising the challenges associated with diagnosing Neisseria gonorrhoeae infection, especially in low-resource areas where the prevalence of infection is highest, scientists at multiple institutes in the US collaborated on a study to develop Cas13a-based assays that could detect N. gonorrhoeae via the porA gene and also predict susceptibility to the antibiotic ciprofloxacin via detection of a single resistance-conferring mutation in the gyrase A (gyrA) gene. The team designed and optimised RNA guides and primer sets for SHERLOCK assays and evaluated their performance using both synthetic DNA and purified N. gonorrhoeae isolates. In an article published yesterday in mSphere, they report the development of Cas13a-based SHERLOCK assays that can detect N. gonorrhoeae and differentiate ciprofloxacin-resistant isolates from ciprofloxacin-susceptible isolates.

Podcasts

• In a recent episode of the Scientific Sense podcast hosted by Gill Eapen, Stephanie Cherqui, Professor in Pediatrics at the University of California San Diego, discusses her research on the molecular characterisation of cystinosis, a metabolic hereditary disease, and ongoing gene therapy strategies that may one day cure this disease. Watch the podcast here. Learn more about the science behind Stephanie's work in our recent interview here.
• In the latest episode of the FLOT.bio podcast, CRISPR Therapeutics' Co-Founder and Chairman Rodger Novak joins host Philip Hemme to chat about bringing the first CRISPR therapeutic (exa-cel) to the market. Listen to that podcast here.

News from CRISPR Medicine News

• On Wednesday, we published an updated clinical trial roundup for the gene-editing therapies in development for sickle cell disease (SCD). There are currently four clinical-stage candidates in development, three of which aim to restore foetal haemoglobin (HbF) expression through gene editing of patient-derived cells, and a single candidate that aims to tackle the root cause of SCD by direcly correcting the disease-causing mutation in patients' own cells. Read the update here.

Huh, heh, wow

• Spider silk stronger than Kevlar spun by an unlikely source. Using a combination of CRISPR and guided egg alterations, researchers at Donghua University in Chinadid created silkworms that can spin silk identical to arachnids. In an article published earlier this week in the journal Matter, they detail how their approach has led to the production of fibres that they claim are already six times tougher than bulletproof Kevlar.