322 Citations found

Jensen S, Charles JR, et al. (2020) A sorghum practical haplotype graph facilitates genome-wide imputation and cost-effective genomic prediction. Plant Genome. doi: 10.1002/tpg2.20009

Researchers developed a database called the Practical Haplotype Graph (PHG) to store haplotype and variant information to manage large genomic datasets for breeding programs. rhAmpSeq amplicon sequencing was used to identify single nucleotide variants (SNPs) which were compared to those predicted by the PHG.

Zou C, Karn A, et al. (2020) Haplotyping the Vitis collinear core genome with rhAmpSeq improves marker transferability in a diverse genus . Nature Communications. doi: 10.1038/s41467-019-14280-1

Agriculture uses molecular markers to accelerate transfer of traits when breeding. The researchers developed a low-cost DNA marker system for the grape genus (Vitis) using the rhAmpSeq amplicon sequencing system.

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
Shapiro J, Iancu O, et al. (2020) Increasing CRISPR efficiency and measuring its specificity in HSPCs using a clinically relevant system. Mol Ther Methods Clin Dev. doi: 10.1016/j.omtm.2020.04.027

This study optimized CRISPR-Cas9 genome editing in an ex vivo system using CD34+ hematopoietic stem and progenitor cells. The researchers created a clear workflow to precisely identify off-target effects using the rhAmpSeq amplicon sequencing system.

Basar R, Daher M, et al. (2020) Large-scale GMP-compliant CRISPR-Cas9–mediated deletion of the glucocorticoid receptor in multivirus-specific T cells. Blood Adv. doi: 10.1182/bloodadvances.2020001977

In this research study, CRISPR-Cas9 was used to delete the glucocorticoid receptor gene in viral specific T cells (VSTs). The gene editing protected the VSTs from lymphocytotoxicity while retaining the glucocorticoid effectiveness against graft-versus-host disease. Off-target edits were characterized by the rhAmpSeq amplicon sequencing system.

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

 

CRISPR-Cas9 was used to correct the sickle mutation in hematopoietic stem and progenitor cells before transplantation into a mouse model of sickle cell disease. GUIDE-seq was used to identify off-target edits and the rhAmpSeq amplicon sequencing system quantified on-target and off-target activity.

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.

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.

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