Kristina M. Cook
  • Research scientist, Kristina Cook
Research fields
  • Cancer biology
Improved HTGTS for CRISPR/Cas9 Off-target Detection
Authors:  Jianhang Yin, Mengzhu Liu, Yang Liu and Jiazhi Hu, date: 05/05/2019, view: 5563, Q&A: 0
Precise genome editing is essential for scientific research and clinical application. At present, linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) is one of most effective methods to evaluate the off-target activity of CRISPR-Cas9, which is based on chromosomal translocation and employs a “bait” DNA double-stranded break (DSB) to capture genome-wide “prey” DNA DSBs. Here, we described an improved HTGTS (iHTGTS) method, in which size-selection beads were used to enhance reaction efficiency and a new primer system was designed to be compatible with Illumina Hiseq sequencing. Compared with LAM-HTGTS, iHTGTS is lower cost and has much higher sensitivity for off-target detection in HEK293T, K562, U2OS and HCT116 cell lines. So we believe that iHTGTS is a powerful method for comprehensively assessing Cas9 off-target effect.
Imaging Higher-order Chromatin Structures in Single Cells Using Stochastic Optical Reconstruction Microscopy
Authors:  Jianquan Xu and Yang Liu, date: 02/05/2019, view: 6430, Q&A: 0
Higher-order chromatin organization shaped by epigenetic modifications influence the chromatin environment and subsequently regulate gene expression. Direct visualization of the higher-order chromatin structure at their epigenomic states is of great importance for understanding chromatin compaction and its subsequent effect on gene expression and various cellular processes. With the recent advances in super-resolution microscopy, the higher-order chromatin structure can now be directly visualized in situ down to the scale of ~30 nm. This protocol provides detailed description of super-resolution imaging of higher-order chromatin structure using stochastic optical reconstruction microscopy (STORM). We discussed fluorescence staining methods of DNA and histone proteins and crucial technical factors to obtain high-quality super-resolution images.
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