Alt-R CRISPR-Cas9 citations

  1. A simple and universal system for gene manipulation in Aspergillus fumigatus: in vitro-assembled Cas9 guide RNA ribonucleoproteins coupled with microhomology repair templates
    Al Abdallah Q, Ge W, Fortwendel JR
    mSphere, 2:e00446–17 (2017)
    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.
  2. DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle
    Mikheikin A, Olsen A, et al
    Nat Commun, 8:1665 (2017)
    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.
  3. Silencing of retrotransposons by SETDB1 inhibits the interferon response in acute myeloid leukemia
    Cuellar TL, Herzner AM, et al
    J Cell Biol, 216:3535–3549 (2017)
    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.
  4. CRISPR-DS: An efficient, low DNA input method for ultra-accurate sequencing
    Nachmanson D, Lian S, et al
    bioRxiv, [Epub]:doi:10.1101/207027 (2017)
    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.
  5. CRISPR-mediated tagging of endogenous proteins with a luminescent peptide
    Schwinn MK, Machleidt T, et al
    ACS Chem Biol, [Epub]:doi: 10.1021/acschembio.7b00549 (2017)
    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.
  6. Dendritic cells but not macrophages sense tumor mitochondrial DNA for cross-priming through signal regulatory protein α signaling
    Xu MM, Pu Y, et al.
    Immunity, 47(2):363–373 (2017)

    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.

  7. An RNAi screen in a novel model of oriented divisions identifies the actin-capping protein Z β as an essential regulator of spindle orientation.
    di Pietro F, Valon L, et al.
    Curr Biol, 27(16):2452–2464 (2017)

    This publication reports a screening effort to uncover novel regulators of vertebrate spindle orientation. The authors describe a method for efficient gene knockout in chick embryos which employs in ovo electroporation of Alt-R CRISPR-Cas9 components (crRNA and tracrRNA).

  8. SCIMP is a transmembrane non-TIR TLR adaptor that promotes proinflammatory cytokine production from macrophages
    Luo L, Bokil N, et al.
    Nat Commun, [Epub]:doi:10.1038/ncomms14133 (2017)

    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.

  9. Superresolution microscopy reveals the three-dimensional organization of meiotic chromosome axes in intact Caenorhabditis elegans tissue
    Kohler S, Wojcik M, et al.
    Proc Natl Acad Sci USA, 114(24):E4734–E4743 (2017)

    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.

  10. Use of RNA-protein complexes for genome editing in non-albicans Candida species
    Grahl N, Demers EG, et al.
    mSphere, 2(3): e00218-17 (2017)
    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.
  11. Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins
    Quadros RM, Miura H, et al.
    Genome Biology, 18(92): (2017)

    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.

  12. Simplified CRISPR tools for efficient genome editing and streamlined protocols for their delivery into mammalian cells and mouse zygotes
    Jacobi AM, Rettig GR, et al.
    Methods, 121–122:16–28 (2017)
    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.
  13. Marker-free coselection for CRISPR-driven genome editing in human cells
    Agudelo D, Duringer A, et al.
    Nature Methods , :[Epub ahead of print] (2017)
    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.
  14. Cas9 ribonucleoprotein delivery via microfluidic cell-deformation chip for human T-Cell genome editing and immunotherapy
    Han X, Liu Z, et al.
    Advanced Biosystems, 1(1):[Epub ahead of print] (2017)

    Here, researchers deliver Cas9 ribonucleoprotein (RNP) to various cell types, including human primary CD4+ T cells, via a novel microfluidic cell deformation-based method.

  15. Gene editing in mouse zygotes using the CRISPR/Cas9 system
    Wefers B, Bashir S, et al.
    Methods, S1046-2023(16):[Epub ahead of print] (2017)
    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.
  16. Insertional mutagenesis by CRISPR/Cas9 ribonucleoprotein gene editing in cells targeted for point mutation repair directed by short single-stranded DNA oligonucleotides
    Rivera-Torres N, Banas K, et al.
    PLoS One, 12(1):e0169350 (2017)
    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.