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CURATOR
A pinboard by
Gilu Francis

Student, Amrita University

PINBOARD SUMMARY

The emergence of CRISPR/Cas9 technique in gene editing has enabled target identification much easier. In the present work, the evaluation of a novel P4 drug molecule has been conducted using CRISPR/Cas9 technique. A potential P4 ‘model drug molecule’ has been designed through octapodial approach using retrometabolic method targeting APC and A3B genes. These genes have been found to be upregulated in colorectal cancer and breast cancer. While transforming P0 drug to P4 drug, the drug action is focused on the DNA. Genes of interest have been separated and taken from an affected organism with very lower level of evolution. The ‘model drug molecule’ has been evaluated invivo using fly model and has been identified to be suppressing APC and thereby can be used as a drug for CRC. Similarly, the molecule has been found to be suppressing A3B gene which is considered as an enzymatic cause of mutations.

6 ITEMS PINNED

Development of a CRISPR/Cas9-mediated gene-editing tool in Streptomyces rimosus.

Abstract: Clustered regularly interspaced short palindromic repeats, associated proteins (CRISPR/Cas), has been developed into a powerful, targeted genome-editing tool in a wide variety of species. Here, we report an extensive investigation of the type II CRISPR/Cas9 system for targeted gene editing in Streptomyces rimosus. S. rimosus is used in the production of the antibiotic oxytetracycline, and its genome differs greatly from other species of the genus Streptomyces in the conserved chromosome terminal and core regions, which is of major production and scientific research value. The genes zwf2 and devB were chosen as target genes, and were edited separately via single-site mutations, double-site mutations and gene fragment disruptions. The single-site mutation guided by sgRNA-1 or sgRNA-2, respectively, involved GG changing to CA, GC changing to AT, and GG changing to CC. The double-site mutations guided by sgRNA-1 and sgRNA-2 included deletions and/or point mutations. Consistently, all mutations occurred in the gRNA sequence regions. Deletion mutations were characterized by the absence of eight bases, including three bases upstream of the PAM (protospacer adjacent motif) sequence, the PAM sequence itself and two bases downstream of the PAM sequence. A mutant (zwf2-devB-) with a high yield of oxytetracycline was successfully obtained, whose oxytetracycline level was increased by 36.8 % compared to the original strain. These results confirm that CRISPR/Cas9 can successfully serve as a useful targeted genome editing system in S. rimosus.

Pub.: 26 Jul '17, Pinned: 27 Jul '17

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer.

Abstract: CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obtained from genetic studies relying on single gene knockouts. We have developed a novel, rapid cloning method for guide RNA (gRNA) concatemers in order to create multi-gene knockouts following a single round of transfection in mouse small intestinal organoids. Our strategy allows for the concatemerization of up to four individual gRNAs into a single vector by performing a single Golden Gate shuffling reaction with annealed gRNA oligos and a pre-designed retroviral vector. This allows either the simultaneous knockout of up to four different genes, or increased knockout efficiency following the targeting of one gene by multiple gRNAs. In this protocol, we show in detail how to efficiently clone multiple gRNAs into the retroviral CRISPR-concatemer vector and how to achieve highly efficient electroporation in intestinal organoids. As an example, we show that simultaneous knockout of two pairs of genes encoding negative regulators of the Wnt signaling pathway (Axin1/2 and Rnf43/Znrf3) renders intestinal organoids resistant to the withdrawal of key growth factors.

Pub.: 27 Jul '17, Pinned: 27 Jul '17