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.
Beltz K, Tsang D, Wan JZ., Rose S, Bao Y, Wang Y, Larkin K, Rupp S, Schrepfer D, Datta K, Gunderson K, Sailor C, Hansen S, Dobosy J, Lewis L, Menezes A, Walder J, Behlke M, Chen CF.
A high-performing and cost-effective SNP genotyping method using rhPCR and universal reporters
Advances in Bioscience and Biotechnology,
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.
Using TruGrade DNA oligos in single-cell antibody sequencing, researchers from Stanford University (Stanford, CA, USA) found that plasmablasts producing Immunoglobulin A (IgA) are elevated in patients suffering from idiopathic pulmonary arterial hypertension (IPAH). They go on to explain how antibody derivatives of the plasmablasts stimulate the production of endothelial cell inflammatory mediators, which may contribute to disease pathogenesis.
The aim of this study was to compare the accuracy, sensitivity, and costs of TaqMan, KASP, and rhAmp SNP genotyping methods in sugar beet (Beta vulgaris L.). rhAmp produced more calls than both TaqMan and KASP, and produced higher signal to NTC data while offering the lowest cost per reaction.
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.
In an example of the evolution of DNA-based nanomachinery, researchers devise 2 anti-parallel DNA origami filaments that undergo step-wise, reversible sliding in opposite directions. Movement is powered by DNA fuels mediated by gold nanocrystals.
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.
PrimeTime Assays using FAM-labeled, ZEN Double-Quenched Probes were used to quantify total mitochondrial DNA in mice.
ZEN Double-Quenched Probes were used to increase the sensitivity of real-time RT-PCR assays designed for analysis of RotaTeq virus. The assays were designed to analyze 5 genotypes (G1, G2, G3, G4, and G6) in the VP7 gene.
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.