Peer-reviewed articles published by IDT scientists. Filter using one or more categories to focus on specific topics.
This report describes the isolation of a Cas9 variant that displays a superior on- to off-target ratio when delivered in RNP format. Robust on-target editing was achieved at therapeutically relevant loci in hard-to-edit primary cells, while overall off-target editing was substantially reduced. The high-fidelity Cas9 enzyme used in the study is now commercially available from IDT as the Alt-R HiFi Cas9 Nuclease V3.
Ferrand J, Croft NP, Pepin G, Diener KR, Wu D, Mangan NE, Pederson J, Behlke MA, Bayball JD, Purcell AW, Ferrero RL, Gantier MP.
The use of CRISPR/Cas9 gene editing to confirm congenic contaminations in host-pathogen interaction studies
Front Cell Infect Microbiol,
Li MA, Amaral PP, Cheung P, Bergmann JH, Kinoshita M, Kalkan T, Ralser M, Robson S, von Meyenn F, Paramor M, Yang F, Chen C, Nichols J, Spector DL, Kouzarides T, He L, Smith A.
A lncRNA fine tunes the dynamics of a cell state transition involving Lin28, let-7 and de novo DNA methylation
Researchers from the University of Washington and TwinStrand Biosciences describe a targeted sequencing approach called CRISPR-DS, which couples a previously described method known as duplex sequencing with CRISPR-Cas9 system for target selection. Alt-R CRISPR-Cas9 RNAs were used in in vitro digestion to fragment input genomic DNA at specified locations, followed by size selection. Compared to standard duplex sequencing approach, CRISPR-DS resulted in 20-fold improvement of on-target rate using only minimal amounts of input DNA.
Choi YJ, Lin CP, Risso D, Chen S, Kim TA, Tan MH, Li JB, Wu Y, Chen C, Xuan Z, Macfarlan T, Peng W, Lloyd KC, Kim SY, Speed TP, He L.
Deficiency of microRNA miR-34a expands cell fate potential in pluripotent stem cells
The authors of this paper describe Easi-CRISPR, a robust and efficient strategy for targeted DNA cassette insertion in mice. The international consortium of 7 research teams injected mouse zygotes with long single-stranded DNA donors (Megamer Single-Stranded DNA Fragments) and pre-assembled Cas9 ribonucleoprotein complexes (Alt-R crRNA, tracrRNA, and Cas9 nuclease), and obtained successful knock-in at 13 loci.
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.
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
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.
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
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,
Begin-Lavallee V, Midavaine E, Dansereau MA, Tetreault P, Longpre JM, Jacobi AM, Rose SD, Behlke MA, Beaudet N, Sarret P.
Functional inhibition of chemokine receptor CCR2 by dicer-substrate-siRNA prevents pain development