Scientific Advances: Synthetic immunomodulation with a CRISPR super-repressor in vivo

The clinical implementation of gene therapy is complicated by a potential immune response to the vectors used for in vivo delivery. In a recent paper by Moghadan et al. CRISPR-mediated transcriptional repression is exploited to dampen the immune response against AAV in vivo.

Reference: Moghadam, F., LeGraw, R., Velazquez, J.J. et al. (2020) Synthetic immunomodulation with a CRISPR super-repressor in vivo. Nat Cell Biol 22, 1143–1154. https://doi.org/10.1038/s41556-020-0563-3

Keywords: Immunosuppression, Gene therapy, CRISPR, Myd88

Questions? Email: crispr@amsterdamumc.nl

CRISPR Dx: Fast, accurate, and point of care COVID-19 diagnostics with Cas12

In light of a pending second COVID-19 wave, an on-site diagnostic test that is fast and accurate could make a real difference in preventing the spread of SARS-CoV-2.

In the following manuscript, uploaded to the medRxiv server before peer review, a multicenter study involving the Amsterdam UMC, Leiden UMC, and OLVG Amsterdam is presented showing that a Cas12-based diagnostic test called DETECTR is as effective as current PCR-based COVID-19 testing. However in comparison to current standards, DETECTR works faster and can be deployed onsite as a point of care test.

Read more:
Pre-Print: Brandsma & Verhagen et al. Rapid, sensitive and specific SARS coronavirus-2 detection: a multi-center comparison between standard qRT-PCR and CRISPR based DETECTR. medRxiv (2020). https://doi.org/10.1101/2020.07.27.20147249

For more information on CRISPR Diagnostics see our news item on the comprehensive review by Van Dongen et al. in Biosensors and Bioelectronics.

Keywords: CRISPR-Dx, COVID-19, DETECTR, Cas12

Questions? Email: crispr@amsterdamumc.nl

Snelle diagnose van het SARS-CoV-2 virus op locatie

Onderzoekers van het Amsterdam UMC hebben in samenwerking met het UMC Leiden en het Onze Lieve Vrouwe Gasthuis Amsterdam een snelle SARS-CoV-2 diagnostische test beoordeeld. Deze snelle COVID-19 test, ook wel DETECTR genoemd werkt op basis van een nieuw detectie principe. Hierbij wordt gebruik gemaakt van een bacterieel eiwit Cas12 dat met behulp van een gids molecuul het SARS-CoV-2 virus specifiek herkend. Uit het werk van de onderzoekers blijkt dat DETECTR even goed werkt als de huidige laboratoriumtesten. Echter een groot voordeel van DETECTR is dat de test sneller een uitslag geeft en dat de deze diagnostiek buiten het laboratorium kan worden toegepast.

Het onderzoek wordt momenteel bestudeerd en beoordeeld door andere wetenschappers. Dus het is nog afwachten wanneer DETECTR in Nederland kan worden toegepast. Met oog op de dreiging van een tweede COVID-19 golf kan een snelle Cas12 Corona test op locatie een bijdrage leveren om verdere verspreiding te beperken.

Lees meer op:
Pre-Print: Brandsma & Verhagen et al. Rapid, sensitive and specific SARS coronavirus-2 detection: a multi-center comparison between standard qRT-PCR and CRISPR based DETECTR. medRxiv (2020). https://doi.org/10.1101/2020.07.27.20147249

Keywords: CRISPR-Dx, COVID-19, DETECTR, Cas12

Vragen? Email: crispr@amsterdamumc.nl

CRISPR Dx: Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities

Want to know more about on-site diagnostics based on CRISPR technology? Van Dongen et al. review the technical aspects CRISPR/Cas technology and (future) applications in point of care diagnostics.

Reference: Jeanne E. van Dongen, Johanna T.W. Berendsen, Renske D.M. Steenbergen, Rob M.F. Wolthuis, Jan C.T. Eijkel, Loes I. Segerink (2020) Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities, Biosensors and Bioelectronics 166: 112445. https://doi.org/10.1016/j.bios.2020.112445.

Keywords: Point-of-care, CRISPR/Cas, DNA sensing, Genomic diagnostic tools, CRISPR sensing, Diagnosis

Questions? Email: crispr@amsterdamumc.nl

New Tools: Base Editing with Minimal Bystander Effects

Up till now the application of base editors was limited due to the fact that other cytosines in the proximity of the intended C->T alteration would also be converted. Now, Lee et al. describe an engineered high-fidelity base editor to overcome previous limitations.

Reference: Lee et al. Single C-to-T substitution using engineered APOBEC3G-nCas9 base editors with minimum genome- and transcriptome-wide off-target effects. Science Advances. (2020)  Vol. 6, no. 29, eaba1773. DOI: 10.1126/sciadv.aba1773

Keywords: base editor, APOBEC

Questions? Email: crispr@amsterdamumc.nl

New Tools: Base Editing of Mitochondrial DNA

Mok et al. describe the discovery of a bacterial toxin (DddA) that acts as a cytidine deaminase. Subsequent adaptation of the enzyme included adding a mitochondrial targeting signal and fusion to TALE proteins to facilitate binding to DNA at a specific location. The mitochondrial base editor can make C–>G and T–>A changes. Although some limitations are described, DddA-derived cytosine base editor (DdCBE) is another powerful tool that originated from the David Liu Lab.

Reference: Mok et al. A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature (2020). doi.org/10.1038/s41586-020-2477-4
See also: Aushev & Herbert. Mitochondrial genome editing gets precise. Nature 583, 521-522 (2020). doi:10.1038/d41586-020-01974-6

Keywords: mitochondria, base editor, cytidine deaminase

Questions? Email: crispr@amsterdamumc.nl

Data in preprint suggest CRISPR genome editing in human embryos drives loss of heterozygosity

Liang et al. report on the outcome of CRISPR/Cas9 gene editing in human embryos in a manuscript titled: “Frequent gene conversions in human embryos induced by double strand breaks”. The preprint was uploaded to the BioRxiv server and has not been peer-reviewed. The authors report that in early stage human embryos Cas9-induced double strand breaks are repaired by gene conversion using an
intact wildtype homolog as a template in up to 40% of targeted embryos. As a result of the expansion of the conversion tracks well beyond the target area, CRISPR/Cas9 activity can result in wide-ranging loss of heterozygosity (LOH). Moreover, the combined action of Cas9, non-homologous end joining (NHEJ), and gene conversion resulted in embryos with identical indel mutations on both target loci. In these experiments Cas9 and gRNA were delivered as ribonucleoproteins (RNPs). Reference: Liang et al. Frequent gene conversions in human embryos induced by double strand breaks. bioRxiv 2020.06.19.162214; https://doi.org/10.1101/2020.06.19.162214

Questions? Email: crispr@amsterdamumc.nl

Which Cas9 nuclease is right for your CRISPR experiment?

In recent years distinct variants have been developed based on the originally adapted nuclease Streptococcus pyogenes Cas9. In a recent issue of Nature Biotechnology, Kim et al. have analyzed the sequence-specific cleavage activity of several Cas9 variants and developed an online prediction tool to help you find the right Cas9 for your experiment.

Reference: Kim et al. Prediction of the sequence-specific cleavage activity of Cas9 variants. Nat Biotechnol (2020). https://doi.org/10.1038/s41587-020-0537-9

Questions? Email: crispr@amsterdamumc.nl