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.
Combining CRISPR-Cas9 genome editing and HiBiT reporter technologies, this study describes an approach to efficiently tag endogenous proteins with a small luminescent peptide. The researchers achieved rapid, high integration efficiency and assay sensitivity via electroporation of a pre-assembled Alt-R Cas9 RNP complex and ssODN templates, ordered as IDT Ultramers. This enabled quantification of protein levels in the mixed population of edited cells without requiring clonal isolation.
This study provides an example of disrupting endogenous gene expression in mouse MC38 cells via electroporation of a pre-assembled Alt-R Cas9 RNP complex. By generating a tumor cell line in which both alleles of transmembrane protein CD47 are knocked out, the researchers show that increased sensing of tumor-derived DNA (achieved by CD47 blockade) primarily occurs in dendritic cells but not in microphages. These findings shed light on the molecular mechanism underlying immune invasion of tumor cells.
Researchers from Virginia Commonwealth University describe a novel CRISPR-Cas9 mediated “nanomapping” approach, which may fill technical gaps that are poorly addressed by existing DNA-mapping techniques. Using Alt-R CRISPR guide RNAs and high-speed atomic force microscopy (HS-AFM), they report successful detection and precise mapping of BCL2-IGH translocations in clinical samples derived from follicular lymphoma patients.
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.
This publication details the process of designing knock-out and knock-in CRISPR experiments for the generation of new mouse mutants. It outlines proper preparation of Cas9 ribonucleoprotein, as well as procedures for delivering the complex to mouse zygotes by way of microinjection and electroporation.
This publication from the laboratory of Dr Eric Kmiec highlights the advantages of using CRISPR-Cas9 ribonucleoprotein for DNA cleavage along with single-stranded DNA oligonucleotides for repair of single base mutations, and examines the mechanism of repair in greater detail.
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.
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.
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.