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.
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.