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.
Researchers at UCSD report creating point mutations and deletions in endogenous genes in C.elegans, using Alt-R crRNA and tracrRNA.
This study provides the first side-by-side comparison of chemically synthesized CRISPR guide RNA versus in vitro transcribed RNA in zebrafish. Synthetic gRNAs provided by IDT yielded efficient targeting and generated both loss of function mutations and precise gene knock-ins in zebrafish embryos.
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 review discusses the utility of CRISPR-Cas9 genome editing systems for creating genetically engineered animals for alcohol research. When comparing commercially available platforms, it specifically highlights Alt-R as the "system of choice" for achieving easy, fast and efficient genome editing in mouse embryos.
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 reports dysregulated blood glucose homeostasis in cave-adapted populations of the Mexican tetra, Astyanax mexicanus, and suggests a beneficial effect of diminished insulin signaling in a nutrient-limited environment. The researchers used the 2-part Alt-R guide RNAs from IDT to introduce cavefish-specific point mutations into zebrafish and study the gain-of-function phenotype.
This study from Genentech describes an optimized method for delivering CRISPR-Cas9 RNP into primary mouse and human T cells. The use of Cas9 RNP transfection overcomes the limitations of all-in-one lentiviral approaches and does not require stimulation of TCR (T cell receptor), thus allowing functional studies of genes involved in T cell activation and differentiation. The researchers achieved highly efficient gene knock-out via nucleofection of a Cas9 RNP formed by Alt-R crRNA and tracrRNA, which largely mitigates the need for selection and clonal isolation.
Researchers from Genentech use IDT Alt-R CRISPR RNAs to efficiently knock out endogenous genes RIPK1 and HOIP in Bone Marrow-Derived Macrophages (BMDM).
Here, researchers deliver Cas9 ribonucleoprotein (RNP) to various cell types, including human primary CD4+ T cells, via a novel microfluidic cell deformation-based method.
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 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).
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.