CMN Weekly (27 August 2021)
• Researchers from South Korea have used prime editing to cure mice of two human diseases - the genetic liver disease, hereditary tyrosinemia, and the genetic eye disease, Leber congenital amaurosis. Prime editing precisely corrected the disease-causing mutations and ameliorated the disease phenotypes. Prime-editing guide RNAs were identified using lentiviral libraries of guide-RNA-encoding sequences paired with the corresponding target sequences. They were delivered by either hydrodynamic injection or an adeno-associated virus vector.
• Researchers in the Netherlands and USA have identified a small molecule that can reproducibly increase the fraction of 1-bp insertions relative to other CRISPR-Cas9 repair outcomes. The small molecule, KU-60019, inhibits the ATM kinase that is otherwise activated by DNA double-strand breaks. The inhibition ensures a relative frequency of 1-bp insertions of over 80% of edited alleles at several native human genomic loci. Thus, KU-60019 might improve the correction efficiency for pathogenic 1-bp deletion variants and add another dimension to precise template-free Cas9-nuclease genome editing.
• Immune responses to the microbial Cas9 protein can have consequences for clinical applications. American researchers have now identified the immunodominant regions of the Staphylococcus aureus Cas9 (SaCas9) protein to avoid that. This was achieved by exposing 209 overlapping peptides that span the entire sequence of SaCas9 to human peripheral blood mononuclear cells (PBMCs) from a cohort of donors with a distribution of Major Histocompatibility Complex (MHC) alleles comparable to that in the North American (NA) population. It turned out that 22 of the 209 SaCas9 peptides were both presented by MHC-II proteins and stimulated CD4+ T-cells.
• Base editors have great potential as genome editing tools, but their application has been hampered by a broad variation in editing efficiencies on different genomic loci. Now, researchers from Switzerland have extensively analysed adenine and cytosine base editors on a library of 28,294 lentivirally integrated genetic sequences. The scientists then established an attention-based deep learning algorithm, BE-DICT, capable of predicting base editing outcomes with high accuracy.
• Repairing double-strand breaks after CRISPR-Cas9 cleavage can lead to undesired large deletions. However, a Chinese research group now shows how these potentially pathogenic events can be controlled. The researchers demonstrate that single-stranded oligodeoxynucleotides carrying short homology can reduce the deletion damage by almost half. In contrast, adeno-associated virus donors with long homology can reduce large deletions by approximately 80%.
• Researchers in Denmark have developed a new non-enzymatic assay for rapid detection of SARS-CoV-2 RNA. The assay uses non-enzymatic, isothermal strand displacement and amplification (NISDA) and can quantify 10 RNA copies/µL with 100% specificity and 96.8-100% sensitivity. The assay depends on a DNA duplex (the initiator) and two DNA molecular beacon structures, and since it does not require enzymes, it is highly robust at room temperature (>1 month).
• Scientists from the USA have determined the cryo-EM structure of Cas12k from cyanobacteria Scytonema hofmanni in complex with a sgRNA and a double-stranded target DNA. The results revealed mechanisms for recognising the GGTT PAM sequence and the structural elements of Cas12k critical for RNA-guided DNA transposition.
• American researchers have developed a protocol for using CRISPR-Cas9 to knock down long noncoding RNAs (lncRNAs). LncRNAs are RNA transcripts greater than 200 nucleotides in length, and they play a crucial role in a myriad of physiological and pathological processes.
• Chinese researchers have developed an ultrasensitive electrochemical biosensor for point-of-care testing (POCT) of pathogenic bacteria without nucleic acid extraction. The method exploits functional DNA aptamers that lock the hairpin of a primer exchange reaction. In the presence of target bacteria, the aptamers leave the hairpin to bind to the bacteria. This initiates the production of ssDNA that unleashes CRISPR/Cas12a activity and leads to an electrochemical signal. As a result, E. coli could be quantified from 10 to 106 CFU·mL−1 with a detection limit of 19 CFU·mL−1.
• Another Chinese research group have developed a CRISPR-Cas9 platform for multiplexed detection of three different HPV strains in a single tube. The platform uses Cas9 and Klenow fragment to produce ssDNA of the target. Bioinspired photonic crystal barcodes then capture the ssDNA, and the colourimetric detection signal is amplified by hybridisation chain reaction. Thus, HPV could be detected and multiplexed with a highly sensitive limit of 0.025 pM.
• Vertex and Arbor Technologies have extended and expanded their previous partnership to enhance efforts in developing ex vivo engineered cell therapies. Vertex will receive rights to use Arbor's technology within therapies for type 1 diabetes, sickle cell disease, beta-thalassemia, and other diseases.
• Editas Medicine has presented new data on its novel gene-editing technology termed SLEEK (SeLection by Essential-gene Exon Knock-in). SLEEK utilises a proprietary Cas12a from Acidaminococcus sp. to obtain almost 100% knock-in frequencies of knock-ins containing a selectable gene.
• Excision BioTherapeutics has received a Martin Delaney Grant from the National Institutes of Health (NIH) of $4.8 million annually for the next five years. The grant is given to a collaboration with Temple University to develop further the company's CRISPR-based HIV DNA ablation therapeutic, EBT-101, as a cure for HIV.
• A new market report by iHealthcareAnalyst estimates the global genome editing market to attain $8.5 billion by 2027, expanding at a CAGR of 10.2% during the forecast period.
• A review in ACS Nano looks at synthetic biology and its potential for developing next-generation adenoviral vectors for gene therapy end editing. Among other benefits, synthetic biology can modify the adenovirus and thus mitigate viral toxicity or provide precise viral tropisms.
• The use of exosomes as a targeted delivery platform of CRISPR-Cas9 for therapeutic genome editing is the focus of another review. Exosomes are natural extracellular carrier vehicles that mediate cell-cell communications. The review looks at recent progress in various strategies to achieve selective delivery to a particular type of cells and efficiently packaging the genome editing tools in the vesicles.
• Another review focuses on the unique features of TALENs and the limitations of CRISPRs, which in some cases makes TALENs a better genome editing tool than CRISPRs. Finally, the authors suggest that the high specificity and sensitivity toward DNA modifications provides TALENs with novel opportunities in therapeutics and synthetic biology.
• CRISPR-Cas advancements in molecular diagnostics and signal readout approaches are the subjects of a review in the Journal of Molecular Diagnostics. The authors discuss CRISPR-Cas detection strategies based on their target hybridisation, cleavage activity, sensor capabilities and signal readout methods, and the recent progressions, challenges and improvement strategies of CRISPR-Cas technology for portable point-of-care diagnostic devices.
• This week’s edition of Nature brings a special Outlook section with ten articles about the sickle-cell disease, and one of them is entitled Can CRISPR cure sickle-cell disease? More than 6 million people live with the disease, and in sub-Saharan Africa, where three-quarters of the patients live, childhood mortality due to sickle cell remains high. An accompanying 10 minute video can be seen on YouTube.
Poseida Therapeutics, Inc.
First Affiliated Hospital of Guangdong Pharmaceutical University