Department of Biochemistry, Carver College of Medicine, University of Iowa, Coralville, IA 52241, USA.
Integrated DNA Technologies, Inc., 1710 Commercial Park, Coralville, IA 52241, USA.
Nucleic Acids Res. 2020 May 21;48(9):5037-5053. doi: 10.1093/nar/gkaa231.
CRISPR RNA-guided endonucleases (RGEs) cut or direct activities to specific genomic loci, yet each has off-target activities that are often unpredictable. We developed a pair of simple in vitro assays to systematically measure the DNA-binding specificity (Spec-seq), catalytic activity specificity (SEAM-seq) and cleavage efficiency of RGEs. By separately quantifying binding and cleavage specificity, Spec/SEAM-seq provides detailed mechanistic insight into off-target activity. Feature-based models generated from Spec/SEAM-seq data for SpCas9 were consistent with previous reports of its in vitro and in vivo specificity, validating the approach. Spec/SEAM-seq is also useful for profiling less-well characterized RGEs. Application to an engineered SpCas9, HiFi-SpCas9, indicated that its enhanced target discrimination can be attributed to cleavage rather than binding specificity. The ortholog ScCas9, on the other hand, derives specificity from binding to an extended PAM. The decreased off-target activity of AsCas12a (Cpf1) appears to be primarily driven by DNA-binding specificity. Finally, we performed the first characterization of CasX specificity, revealing an all-or-nothing mechanism where mismatches can be bound, but not cleaved. Together, these applications establish Spec/SEAM-seq as an accessible method to rapidly and reliably evaluate the specificity of RGEs, Cas::gRNA pairs, and gain insight into the mechanism and thermodynamics of target discrimination.
CRISPR RNA 引导的内切酶（RGEs）可将活性切割或引导至特定的基因组位点，但每种酶都具有不可预测的脱靶活性。我们开发了一对简单的体外测定法，可系统地测量 RGE 的 DNA 结合特异性（Spec-seq）、催化活性特异性（SEAM-seq）和切割效率。通过分别定量结合和切割特异性，Spec/SEAM-seq 可深入了解脱靶活性的机制。基于 Spec/SEAM-seq 数据为 SpCas9 生成的基于特征的模型与 SpCas9 体外和体内特异性的先前报道一致，验证了该方法。Spec/SEAM-seq 也可用于分析特征研究较少的 RGE。该方法在工程化的 SpCas9（HiFi-SpCas9）上的应用表明，其增强的靶标区分能力归因于切割而非结合特异性。另一方面，ScCas9 的特异性源于与扩展的 PAM 结合。AsCas12a（Cpf1）脱靶活性降低似乎主要是由 DNA 结合特异性驱动的。最后，我们首次对 CasX 的特异性进行了表征，揭示了一种全有或全无的机制，其中错配可以结合，但不能切割。总之，这些应用将 Spec/SEAM-seq 确立为一种快速可靠地评估 RGE、Cas::gRNA 对以及深入了解靶标区分的机制和热力学的方法。
