Raw data for "Efficient in vivo labeling of endogenous proteins with SMART delineates retina cellular and synaptic organization" paper.

A key application of CRISPR/Cas9-based genomic editing is modification of genes to introduce engineered sequences. However, the editing flexibility is severely constrained by the requirement for targeting sites in proximity to the desired modification site, which makes many modifications intractable. Here, we develop a strategy that overcomes this key limitation to allow CRISPR-based editing at any position with high efficiency. It relies on reconstructing the targeted gene using Silently Mutate And Repair Template (SMART) where we mutate the gap sequence in the repair template to prevent its base pairing with the target DNA while maintaining the same amino acid coding. Using vertebrate retina as a neuronal model system we document the application of SMART editing for labeling endogenous proteins in vivo with high efficiency. We show that SMART editing allows us to access numerous cell types in the retina and address fundamental cell biological questions pertaining to its organization. We propose that this approach will facilitate functional genomic studies in a wide range of systems and increase the precision of corrective gene therapies.

基于CRISPR/Cas9的基因组编辑的核心应用之一，是对基因进行修饰以引入工程化序列。然而，该技术的编辑灵活性受到严重限制——其要求在目标修饰位点附近设计靶向位点，致使诸多修饰难以实现。本研究开发了一种策略，可克服这一核心局限，实现任意位点的高效CRISPR介导编辑。该策略借助静默突变与修复模板（Silently Mutate And Repair Template, SMART）实现靶向基因的重构：我们对修复模板中的间隙序列进行突变，使其无法与靶DNA形成碱基配对，同时保持原有氨基酸编码信息不变。本研究以脊椎动物视网膜作为神经元模型系统，验证了SMART编辑可高效在体内标记内源蛋白。研究表明，SMART编辑可靶向视网膜内的多种细胞类型，助力解决与其组织结构相关的核心细胞生物学问题。我们认为，该方法将推动多系统的功能基因组学研究，并提升校正型基因治疗的精准性。