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.
This study describes EEZy (Easy Electroporation of Zygotes), an easily adaptable electroporation approach for introducing CRISPR/Cas9-mediated genome editing in C57BL/6 mice, using Alt-R CRIPSR-Cas9 ribonucleoprotein (RNP) and the widely available Bio-Rad GenePulser Xcell electroporator. The authors demonstrate that RNP delivery of CRISPR-Cas9 components comprising of paired crRNA:tracrRNA complexes yields highly efficient editing in up to 100% of the living offspring and has minimal impact on embryo viability. Notably, in electroporation, Alt-R bipartite RNAs show significantly less embryo toxicity compared to sgRNAs generated by in vitro transcription.
This paper describes a rapid, cheap, and accessible analytic tool called TIDER (Tracking of Insertions, Deletions and Recombination events) that can be used to quantify incorporation frequency CRISPR-directed mutations. TIDER is derived from the widely used TIDE and the researchers here used the RNP CRISPR approach from IDT.
This study reports a DNA-free genome editing method in potato via delivery of Alt-R CRISPR-Cas9 ribonucleoprotein (RNP) to potato protoplasts. The authors demonstrate that RNP delivery of CRISPR-Cas9 components comprising synthetic RNA yields higher frequencies of transgene-free mutated lines compared to using in vitro transcribed RNA or plasmid DNA delivery. Therefore, they propose using RNP with synthetic RNAs for CRISPR in potato plants to simplify analysis and selection of commercial crop lines.
This study describes a robust and simple-to-perform method called improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD), which delivers CRISPR RNP to E0.7 embryos via in situ electroporation.
This study shows that in vitro-assembled, dual Alt-R Cas9 RNPs coupled with microhomology repair templates enable efficient gene manipulation in different genetic backgrounds of A. fumigatus.
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.
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.
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.
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.
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.