High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding
High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding
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DOI：
10.1073/pnas.1700557114
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发表时间：
2017-05-23
影响因子：
11.1
通讯作者：
Greenleaf, William J.
中科院分区：
文献类型：
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作者：
Boyle, Evan A.;Andreasson, Johan O. L.;Greenleaf, William J.
细菌适应性免疫系统CRISPR-Cas9已被用作编辑基因组、控制基因表达和可视化遗传位点的通用工具。为了分析Cas9快速特异性结合DNA的能力，我们生成了多个潜在结合伙伴文库，用于测量核酸酶死亡Cas9 （dCas9）相互作用的动力学。使用大规模平行方法定量高通量测序流细胞上蛋白质- dna相互作用，我们全面评估了种子和更远端核苷酸中向导RNA （gRNA）和靶核苷酸组合错配的影响，以及原间隔器邻近基序（PAM）的破坏。我们报告了pam -远端错配的两个后果：在长时间尺度上逆转dCas9的结合，以及当存在其他grna靶标错配时关联动力学的协同变化。总之，这些观察结果支持Cas9特异性模型，其中pam -远端碱基的gRNA-DNA错配调节了决定关联和解离速率的不同生物物理参数。我们提出的方法解耦了Cas9-DNA相互作用的动力学和热力学性质，拓宽了研究脱靶结合行为的工具。
The bacterial adaptive immune system CRISPR-Cas9 has been appropriated as a versatile tool for editing genomes, controlling gene expression, and visualizing genetic loci. To analyze Cas9's ability to bind DNA rapidly and specifically, we generated multiple libraries of potential binding partners for measuring the kinetics of nuclease-dead Cas9 (dCas9) interactions. Using a massively parallel method to quantify protein-DNA interactions on a high-throughput sequencing flow cell, we comprehensively assess the effects of combinatorial mismatches between guide RNA (gRNA) and target nucleotides, both in the seed and in more distal nucleotides, plus disruption of the protospacer adjacent motif (PAM). We report two consequences of PAM-distal mismatches: reversal of dCas9 binding at long time scales, and synergistic changes in association kinetics when other gRNA-target mismatches are present. Together, these observations support a model for Cas9 specificity wherein gRNA-DNA mismatches at PAM-distal bases modulate different biophysical parameters that determine association and dissociation rates. The methods we present decouple aspects of kinetic and thermodynamic properties of the Cas9-DNA interaction and broaden the toolkit for investigating off-target binding behavior.