Researchers from the Doyon laboratory in Quebec describe a method for multiplexing CRISPR guide RNAs, which enables a coselection strategy that can be used to enrich populations of successfully-edited cells following non-homolgous end joining or homology-directed repair.
In order to operate the International Space Station (ISS) National Laboratory more like an Earth-based lab, NASA has developed a molecular biology suite for microgravity conditions called WetLab-2. WetLab-2 is composed of tools, reagents, and methods, which allow on-orbit processing of biological samples and real-time gene expression analysis in space.
This paper describes the results from the WetLab-2 validation experiments. Specifically, qPCR was performed on a concentration series of DNA calibration standards, and RT-qPCR with ZEN Double-Quenched Probes was conducted on RNA that had been extracted and purified (on-orbit) from frozen E. Coli and mouse liver tissue.
Using DNA/RNA hybrid oligos from IDT, a group of researchers at Johns Hopkins University have developed a new technique for analyzing RNA in clinical FFPE specimens that offers numerous advantages over traditional methods.
This study provides an example of downregulating endogenous gene expression in mammalian cells via the use of IDT predesigned Dicer-substrate siRNAs (DsiRNAs). By effectively knocking down the expression level of endogenous gene PEBP1 in both human and mouse cell lines, the researchers show that lowered expression of PEBP1 is associated with decreased sensitivity to ferroptosis, a form of programmed cell death that is pathogenic to several acute and chronic diseases.
Flenker KS, Burghardt EL, Dutta N, Burns WJ, Grover JM, Kenkel EJ, Weaver TM, Mills J, Kim H, Huang L, Owczarzy R, Musselman CA, Behlke MA, Ford B, McNamara JO 2nd.
Rapid detection of urinary tract infections via bacterial nuclease activity
This publication from the laboratories of Dr Jennifer Stow and Matthew Sweet at the University of Queensland details the generation of gene knock-out in mouse cell line RAW264.7. In their experiments, the group achieved successful genome editing by administering pre-assembled RNP complexes (Alt-R crRNA, tracrRNA, and Cas9 nuclease) via lipofection.
This study from Genentech provides an example of knocking out endogenous genes in a human THP-1 cell line that constitutively expresses S.pyogenes Cas9 protein, via nucleofection of a pre-assembled Alt-R crRNA and tracrRNA duplex.
Jacobi AM, Rettig GR, Turk R, Collingwood MA, Zeiner SA, Quadros RM, Harms DW, Bonthuis PJ, Gregg C, Ohtsuka M, Gurumurthy CB, Behlke MA.
Simplified CRISPR tools for efficient genome editing and streamlined protocols for their delivery into mammalian cells and mouse zygotes
Research scientists from IDT and the Gurumurthy lab (University of Nebraska Medical Center) describe methods for genome editing with ribonucleoprotein RNP complexes, which contain chemically-modified, synthetic guide RNAs and recombinant Cas9 protein. RNP delivery methods are described for lipofection and electroporation in mammalian cells, as well as microinjection in murine zygotes, either with or without addition of single-stranded HDR template DNA.
Combining superresolution microscopy with CRISPR-Cas9 genome editing, researchers from Abby Dernburg’s group at UC Berkeley describe a method for building three-dimensional models of the synapsed chromosome axis in C. elegans. Using Alt-R ribonucleoprotein RNP complexes rather than conventional expression plasmids, they report 50-fold improvement of editing efficiency, as measured by the percentage of F1 progeny positive for co-injection markers.
Researchers at the Geisel School of Medicine at Dartmouth describe an expression-free method of CRISPR-Cas9 genome editing in three non-albicans Candida species using Alt-R Cas9 nuclease and guide RNAs. In this publication, Grahl et al. describe the challenges of using exogenously-expressed Cas9 and gRNAs in these species, and how the use of RNA-protein complexes (ribonucleoprotein) can be used to overcome this obstacle, expanding the potential for CRISPR-Cas9 genome editing to a wider range of fungi species.
The authors create DNA origami nanostructures that undergo light-induced conformational changes. Two linked 14-helix origami bundles form a chiral object with a tunable angle. A photo-responsive, azobenzene-modified DNA segment is added to the template and upon illumination, is converted to a cis-form, altering the angle of the bound origami bundles.
The authors identify a previously unknown stimulatory mechanism (through RAD51 paralog proteins) for homologous recombination--filament remodeling. They use DNA origami nanostructures to elucidate the mechanism by which RAD51 paralogs enhance homologous recombination.
Robertson KA, Hsieh WY, Forster T, Blanc M, Lu H, Crick PJ, Yutuc E, Watterson S, Martin K, Griffiths SJ, Enright AJ, Yamamoto M, Pradeepa MM, Lennox KA, Behlke MA, Talbot S, Haas J, Dölken L, Griffiths WJ, Wang Y, Angulo A, Ghazal P.
An interferon regulated microRNA provides broad cell-intrinsic antiviral immunity through multihit host-directed targeting of the sterol pathway
The authors used gBlocks® Gene Fragments to generate a standard curve for their qPCR analysis.
Pépin G, Ferrand J, Höning K, Jayasekara WS, Cain JE, Behlke MA, Gough DJ, G Williams BR, Hornung V, Gantier MP.
Cre-dependent DNA recombination activates a STING-dependent innate immune response
Nucleic Acids Res,
This research team used candidate aptamers in an enzyme-linked aptamer sorbent assay (ELASA) to detect human insulin-like growth factor-I, a biomarker for recombinant human growth hormone, used in athletic doping.