Rarely acquired type II-A CRISPR-Cas spacers mediate anti-viral immunity through the targeting of a non-canonical PAM sequence.
Rarely acquired type II-A CRISPR-Cas spacers mediate anti-viral immunity through the targeting of a non-canonical PAM sequence.
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DOI：
10.1093/nar/gkad501
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发表时间：
2023-08-11
影响因子：
14.9
通讯作者：
中科院分区：
文献类型：
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化脓性链球菌II-A型CRISPR-Cas系统通过从入侵病毒基因组获得短DNA序列（称为间隔区）来提供适应性免疫。间隔区被转录成与病毒基因组区域匹配的短RNA向导，随后是保守的NGG DNA基序，称为PAM。这些RNA向导反过来又被Cas9核酸酶用来发现和破坏病毒基因组内的互补DNA靶标。虽然在噬菌体感染后存活的细菌群体中存在的大多数间隔区靶向侧接NGG序列的原型间隔区，但有一小部分靶向非典型PAM。这些间隔区是否来源于偶然获得的噬菌体序列和/或提供有效的防御是未知的。在这里，我们发现它们中的许多匹配侧接NAGG PAM的噬菌体靶区域。尽管在细菌群体中几乎不存在，但NAGG间隔区在体内提供了实质性的免疫力，并产生了支持Cas9在体外进行稳健DNA切割的RNA向导;这两种活性都与靶向序列的间隔区相当，随后是典型的AGG PAM。相比之下，采集实验表明，NAGG间隔区在非常低的频率下采集。因此，我们得出结论，对这些序列的歧视发生在宿主的免疫过程中。我们的结果揭示了在II-A型CRISPR-Cas免疫应答的间隔区获取和靶向阶段期间PAM识别的意外差异。
The Streptococcus pyogenes type II-A CRISPR-Cas systems provides adaptive immunity through the acquisition of short DNA sequences from invading viral genomes, called spacers. Spacers are transcribed into short RNA guides that match regions of the viral genome followed by a conserved NGG DNA motif, known as the PAM. These RNA guides, in turn, are used by the Cas9 nuclease to find and destroy complementary DNA targets within the viral genome. While most of the spacers present in bacterial populations that survive phage infection target protospacers flanked by NGG sequences, there is a small fraction that target non-canonical PAMs. Whether these spacers originate through accidental acquisition of phage sequences and/or provide efficient defense is unknown. Here we found that many of them match phage target regions flanked by an NAGG PAM. Despite being scarcely present in bacterial populations, NAGG spacers provide substantial immunity in vivo and generate RNA guides that support robust DNA cleavage by Cas9 in vitro; with both activities comparable to spacers that target sequences followed by the canonical AGG PAM. In contrast, acquisition experiments showed that NAGG spacers are acquired at very low frequencies. We therefore conclude that discrimination against these sequences occurs during immunization of the host. Our results reveal unexpected differences in PAM recognition during the spacer acquisition and targeting stages of the type II-A CRISPR-Cas immune response.
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影响因子：
46.9
作者：
Bikard, David;Euler, Chad W.;Jiang, Wenyan;Nussenzweig, Philip M.;Goldberg, Gregory W.;Duportet, Xavier;Fischetti, Vincent A.;Marraffini, Luciano A.
通讯作者：
Marraffini, Luciano A.
影响因子：
14.9
作者：
Jakhanwal S;Cress BF;Maguin P;Lobba MJ;Marraffini LA;Doudna JA
通讯作者：
Doudna JA
影响因子：
64.8
作者：
Anders, Carolin;Niewoehner, Ole;Duerst, Alessia;Jinek, Martin
通讯作者：
Jinek, Martin
影响因子：
64.8
作者：
Goldberg, Gregory W.;Jiang, Wenyan;Bikard, David;Marraffini, Luciano A.
通讯作者：
Marraffini, Luciano A.
影响因子：
16
作者：
Leenay RT;Maksimchuk KR;Slotkowski RA;Agrawal RN;Gomaa AA;Briner AE;Barrangou R;Beisel CL
通讯作者：
Beisel CL