316 Citations found

1. Sebesta J, Peebles C . (2020) ) Improving heterologous protein expression in Synechocystis sp. PCC 6803 for alpha-bisabolene production. Metabol Eng Comm. doi: 10.1016/j.mec.2019.e00117
Wegner M, Diehl V, et al.. (2019) Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome. eLife. doi: http://10.7554/eLife.42549

 

Paix A, Rasoloson D, Folkmann A, Seydoux G. (2019) Rapid tagging of human proteins with fluorescent reporters by genome engineering using double-stranded DNA donors. Curr Protoc Mol Biol. doi: 10.1002/cpmb.102
Blazejewski T, Ho HI, Wang HH. (2019) Synthetic sequence entanglement augments stability and containment of genetic information in cells. Science. doi: 10.1126/science.aav5477

Constraining Adaptive Mutations using Engineered Overlapping Sequences (CAMEOS) is a new computational platform that uses overlapping sequences to prevent protein function disruption. Read how Blazejewski, et al, used gBlocks Gene Fragments and tested their algorithm to ensure that synthetic protein encoding is safeguarded against mutations.

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. (2018) A high-performing and cost-effective SNP genotyping method using rhPCR and universal reporters. Advances in Bioscience and Biotechnology, 9 : 497–512.
Kim KW, Tang NH, et al. (2018) A neuronal piRNA pathway inhibits axon regeneration in C. elegans. Neuron, 97 : 1–9.

Researchers at UCSD report creating point mutations and deletions in endogenous genes in C.elegans, using Alt-R crRNA and tracrRNA.

2. Razeq FM, Jurak E, et al. . (2018) A novel acetyl xylan esterase enabling complete deacetylation of substituted xylans.. Biotechnol Biofuels. doi: 10.1186/s13068-018-1074-3.
DiNapoli SE, Martinze-McFaline R, et al. (2018) A total synthetic approach to CRISPR/Cas9 genome editing and homology directed repair. bioRxiv. doi: 10.1101/359984

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.

Tröder SE, Eber LK, et al. (2018) An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes. PLoS One, 13 (5) : e0196891.

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.

Schubert MS, Cedrone E, Neun B, Behlke MA, Dobrovolskaia MA. (2018) Chemical modification of CRISPR gRNAs eliminate type I interferon responses in human peripheral blood mononuclear cells. J Cytokine Biol, 3 : 1–7.

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.

Broccanello C, Chiodi C, et al. (2018) Comparison of three PCR-based assays for SNP genotyping in plants. Plant Methods, 14 : 28.

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.

Brinkman EK, Kousholt AN, et al. (2018) Easy quantification of template-directed CRISPR/Cas9 editing. Nucleic Acids Res. doi: 10.1093/nar/gky164

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.

Gregg E. Homanics. (2018) Gene edited CRISPy critters for alcohol research. Alcohol. doi: 10.1016/j.alcohol.2018.03.001

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.

Andersson M, Turesson H, et al. (2018) Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery. Physiol Plant. doi: 10.1111/ppl.12731

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.

Urban MJ, Both S, et al. (2018) Gold nanocrystal-mediated sliding of doublet DNA origami filaments.. Nature Comm. doi: http://doi.org/10.1038/s41467-018-03882-w

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.

Ohtsuka M, Sato M, et al. (2018) i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases. Genome Biol, 19 : 25.

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.

Riddle MR, Aspiras AC, et al. (2018) Insulin resistance in cavefish as an adaptation to a nutrient-limited environment. Nature, 555 : 647–651.

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.

Bacman SR, Kauppila JHK, et al. (2018) MitoTALEN reduces mutant mtDNA load and restores tRNA levels in a mouse model of heteroplasmic mtDNA mutation. Nat Med. doi: 10.1038/s41591-018-0166-8

PrimeTime Assays using FAM-labeled, ZEN Double-Quenched Probes were used to quantify total mitochondrial DNA in mice.

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