CRISPR-Cas systems offer versatile technologies for genome engineering, yet their implementation has been outpaced by ongoing discoveries of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation (TXTL) systems to vastly improve the speed and scalability of CRISPR characterization and validation. TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression—all without protein purification or live cells. We used TXTL to measure the dynamics of DNA cleavage and gene repression for single- and multi-effector CRISPR nucleases, predict gene repression strength in E. coli, determine the specificities of 24 diverse anti-CRISPR proteins, and develop a fast and scalable screen for protospacer-adjacent motifs that was successfully applied to five uncharacterized Cpf1 nucleases. These examples underscore how TXTL can facilitate the characterization and application of CRISPR technologies across their many uses.

CRISPR-Cas 系统为基因组工程提供了多种技术，但其实施速度却落后于新 Cas 核酸酶和抗 CRISPR 蛋白的不断发现。在这里，我们展示了使用大肠杆菌无细胞转录翻译 (TXTL) 系统来极大提高 CRISPR 表征和验证的速度和可扩展性。 TXTL 可以通过添加的质粒和线性 DNA 表达活性 CRISPR 机制，并且 TXTL 可以输出 DNA 切割和基因抑制的定量动态 - 所有这些都无需蛋白质纯化或活细胞。我们使用 TXTL 测量单效应和多效应 CRISPR 核酸酶的 DNA 切割和基因抑制动态，预测大肠杆菌中的基因抑制强度，确定 24 种不同抗 CRISPR 蛋白的特异性，并开发快速且可扩展的筛选用于原型间隔子相邻基序，已成功应用于五种未表征的 Cpf1 核酸酶。这些例子强调了 TXTL 如何促进 CRISPR 技术在多种用途中的表征和应用。