Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems.
Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems.
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DOI：
10.1016/j.molcel.2016.02.031
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发表时间：
2016-04-07
期刊：
影响因子：
16
通讯作者：
Beisel CL
中科院分区：
文献类型：
--
作者：
Leenay RT;Maksimchuk KR;Slotkowski RA;Agrawal RN;Gomaa AA;Briner AE;Barrangou R;Beisel CL
原核生物中的CRISPR-Cas适应性免疫系统拥有多种蛋白质家族和作用机制，其中大多数系统依赖于原间隔区邻近基序（PAM）进行DNA靶标识别。在这里，我们开发了一种在体内，积极的，可调屏幕称为PAM-SCANR（PAM屏幕实现NOT门抑制），以阐明功能PAM以及交互式可视化方案称为PAM轮传达个别PAM序列和它们的活动。PAM-SCANR和PAM轮鉴定了已知的功能性PAM，同时揭示了耐盐芽孢杆菌I-C（Cascade）、大肠杆菌I-E（Cascade）、嗜热链球菌II-A CRISPR 1（Cas9）和新杀弗朗西斯菌V-A（Cpf 1）系统的复杂序列-活性景观。PAM轮也很容易适用于现有的高通量筛选，并获得了对SpyCas 9和SauCas 9 PAM多样性的见解。这些工具提供了阐明和可视化功能PAM的强大手段，以加速我们理解和利用自然界中众多CRISPR-Cas系统的能力。
CRISPR-Cas adaptive immune systems in prokaryotes boast a diversity of protein families and mechanisms of action, where most systems rely on protospacer-adjacent motifs (PAMs) for DNA target recognition. Here, we developed an in vivo, positive, and tunable screen termed PAM-SCANR (PAM screen achieved by NOT-gate repression) to elucidate functional PAMs as well as an interactive visualization scheme termed the PAM wheel to convey individual PAM sequences and their activities. PAM-SCANR and the PAM wheel identified known functional PAMs while revealing complex sequence-activity landscapes for the Bacillus halodurans I-C (Cascade), Escherichia coli I-E (Cascade), Streptococcus thermophilus II-A CRISPR1 (Cas9), and Francisella novicida V-A (Cpf1) systems. The PAM wheel was also readily applicable to existing high-throughput screens and garnered insights into SpyCas9 and SauCas9 PAM diversity. These tools offer powerful means of elucidating and visualizing functional PAMs toward accelerating our ability to understand and exploit the multitude of CRISPR-Cas systems in nature.