Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System.

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).

Discovery of genes and proteins possibly regulating mean wool fibre diameter using cDNA microarray and proteomic approaches.

Wool fibre diameter (WFD) is one of the wool traits with higher economic impact. However, the main genes specifically regulating WFD remain unidentified. In this current work we have used Agilent Sheep Gene Expression Microarray and proteomic technology to investigate the gene expression patterns of body side skin, bearing more wool, in Aohan fine wool sheep, a Chinese indigenous breed, and compared them with that of small tail Han sheep, a sheep bread with coarse wool. Microarray analyses showed that most of the genes likely determining wool diameter could be classified into a few categories, including immune response, regulation of receptor binding and growth factor activity. Certain gene families might play a role in hair growth regulation. These include growth factors, immune cytokines, solute carrier families, cellular respiration and glucose transport amongst others. Proteomic analyses also identified scores of differentially expressed proteins.