Department of Biology, Emmanuel College, Boston, United States of America.
Department of Mathematics, Emmanuel College, Boston, United States of America.
PLoS One. 2022 May 6;17(5):e0268099. doi: 10.1371/journal.pone.0268099. eCollection 2022.
A protein roadblock forms when a protein binds DNA and hinders translocation of other DNA binding proteins. These roadblocks can have significant effects on gene expression and regulation as well as DNA binding. Experimental methods for studying the effects of such roadblocks often target endogenous sites or introduce non-variable specific sites into DNAs to create binding sites for artificially introduced protein roadblocks. In this work, we describe a method to create programmable roadblocks using dCas9, a cleavage deficient mutant of the CRISPR effector nuclease Cas9. The programmability allows us to custom design target sites in a synthetic gene intended for in vitro studies. These target sites can be coded with multivalency-in our case, internal restriction sites which can be used in validation studies to verify complete binding of the roadblock. We provide full protocols and sequences and demonstrate how to use the internal restriction sites to verify complete binding of the roadblock. We also provide example results of the effect of DNA roadblocks on the translocation of the restriction endonuclease NdeI, which searches for its cognate site using one dimensional diffusion along DNA.
当蛋白质结合 DNA 并阻碍其他 DNA 结合蛋白的易位时，就会形成蛋白质路障。这些路障会对基因表达和调控以及 DNA 结合产生重大影响。研究这些路障影响的实验方法通常针对内源性位点，或在 DNA 中引入非可变特定位点，以创建用于人为引入蛋白质路障的结合位点。在这项工作中，我们描述了一种使用 dCas9（CRISPR 效应核酸酶 Cas9 的无切割突变体）创建可编程路障的方法。可编程性允许我们在用于体外研究的合成基因中自定义设计目标位点。这些目标位点可以用多价性编码-在我们的情况下，内部限制位点，可以在验证研究中用于验证路障的完全结合。我们提供完整的协议和序列，并演示如何使用内部限制位点来验证路障的完全结合。我们还提供了 DNA 路障对限制内切酶 NdeI 易位影响的示例结果，该酶使用沿 DNA 的一维扩散搜索其同源位点。
