Guan Juan, Liu Harrison, Shi Xiaoyu, Feng Siyu, Huang Bo
University of California, San Francisco, San Francisco, California.
University of California, San Francisco, San Francisco, California.
Biophys J. 2017 Mar 28;112(6):1077-1084. doi: 10.1016/j.bpj.2017.01.032.
Live imaging of genome has offered important insights into the dynamics of the genome organization and gene expression. The demand to image simultaneously multiple genomic loci has prompted a flurry of exciting advances in multicolor CRISPR imaging, although color-based multiplexing is limited by the need for spectrally distinct fluorophores. Here we introduce an approach to achieve highly multiplexed live recording via correlative CRISPR imaging and sequential DNA fluorescence in situ hybridization (FISH). This approach first performs one-color live imaging of multiple genomic loci and then uses sequential rounds of DNA FISH to determine the loci identity. We have optimized the FISH protocol so that each round is complete in 1 min, demonstrating the identification of seven genomic elements and the capability to sustain reversible staining and washing for up to 20 rounds. We have also developed a correlation-based algorithm to faithfully register live and FISH images. Our approach keeps the rest of the color palette open to image other cellular phenomena of interest, as demonstrated by our simultaneous live imaging of genomic loci together with a cell cycle reporter. Furthermore, the algorithm to register faithfully between live and fixed imaging is directly transferrable to other systems such as multiplex RNA imaging with RNA-FISH and multiplex protein imaging with antibody-staining.
基因组的实时成像为深入了解基因组组织和基因表达的动态变化提供了重要见解。同时对多个基因组位点进行成像的需求推动了多色CRISPR成像技术取得了一系列令人兴奋的进展，尽管基于颜色的多重检测受到对光谱上不同荧光团需求的限制。在此，我们介绍一种通过相关CRISPR成像和顺序DNA荧光原位杂交（FISH）实现高度多重实时记录的方法。该方法首先对多个基因组位点进行单色实时成像，然后使用多轮顺序DNA FISH来确定位点身份。我们优化了FISH方案，使每一轮在1分钟内完成，证明了可识别七个基因组元件，并具备进行多达20轮可逆染色和洗涤的能力。我们还开发了一种基于相关性的算法，用于准确配准实时图像和FISH图像。我们的方法使其余的颜色调色板可用于对其他感兴趣的细胞现象进行成像，如我们同时对基因组位点和细胞周期报告基因进行实时成像所展示的那样。此外，在实时成像和固定成像之间进行准确配准的算法可直接应用于其他系统，如使用RNA-FISH的多重RNA成像和使用抗体染色的多重蛋白质成像。
