Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9.
Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9.
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DOI：
10.1128/mbio.00648-15
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发表时间：
2015-06-16
期刊：
影响因子：
6.4
通讯作者：
Bushman FD
中科院分区：
文献类型：
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作者：
Bryson AL;Hwang Y;Sherrill-Mix S;Wu GD;Lewis JD;Black L;Clark TA;Bushman FD
尾状噬菌体的基因组dna通常被化学基团的附着修饰。已知某些形式的DNA修饰可以保护噬菌体DNA免受限制性内切酶的切割，但其他形式的功能未知。最近，CRISPR-Cas核酸酶复合物被证明可以通过rna引导的靶标识别介导细菌适应性免疫，这就提出了噬菌体DNA修饰是否也可以阻断CRISPR-Cas9的攻击的问题。我们研究了噬菌体T4作为模型系统，其中胞嘧啶被葡萄糖-羟甲基胞嘧啶（glc-HMC）取代。我们首先通过单分子DNA测序和酶探针量化了T4 DNA共价修饰的程度和分布。然后，我们设计了靶向T4的CRISPR间隔序列，发现含有glc-HMC的野生型T4对CRISPR- cas9的攻击不敏感，而未经修饰的胞嘧啶突变体则敏感。HMC噬菌体仅表现中等敏感性。虽然这项工作正在进行中，但另一组报告了大量工程化的CRISRP-Cas9复合物的例子，实际上可以克服T4 DNA修饰的影响，这表明修饰可以抑制但并不总是完全阻断攻击。细菌最近被发现具有一种适应性免疫，即CRISPR-Cas系统，它利用核酸配对来识别和切割噬菌体等入侵者的基因组DNA。对尾状噬菌体的历史研究表明，噬菌体DNA通常通过大化学基团的共价附着来修饰。在这里，我们证明了T4噬菌体中的DNA修饰抑制了CRISPR-Cas9系统的攻击。这一发现提供了宿主-病毒竞争机制的见解，也提供了一套新的工具，可能有助于在基因组工程应用中调节CRISPR-Cas9的活性。
The genomic DNAs of tailed bacteriophages are commonly modified by the attachment of chemical groups. Some forms of DNA modification are known to protect phage DNA from cleavage by restriction enzymes, but others are of unknown function. Recently, the CRISPR-Cas nuclease complexes were shown to mediate bacterial adaptive immunity by RNA-guided target recognition, raising the question of whether phage DNA modifications may also block attack by CRISPR-Cas9. We investigated phage T4 as a model system, where cytosine is replaced with glucosyl-hydroxymethylcytosine (glc-HMC). We first quantified the extent and distribution of covalent modifications in T4 DNA by single-molecule DNA sequencing and enzymatic probing. We then designed CRISPR spacer sequences targeting T4 and found that wild-type T4 containing glc-HMC was insensitive to attack by CRISPR-Cas9 but mutants with unmodified cytosine were sensitive. Phage with HMC showed only intermediate sensitivity. While this work was in progress, another group reported examples of heavily engineered CRISRP-Cas9 complexes that could, in fact, overcome the effects of T4 DNA modification, indicating that modifications can inhibit but do not always fully block attack. Bacteria were recently found to have a form of adaptive immunity, the CRISPR-Cas systems, which use nucleic acid pairing to recognize and cleave genomic DNA of invaders such as bacteriophage. Historic work with tailed phages has shown that phage DNA is often modified by covalent attachment of large chemical groups. Here we demonstrate that DNA modification in phage T4 inhibits attack by the CRISPR-Cas9 system. This finding provides insight into mechanisms of host-virus competition and also a new set of tools that may be useful in modulating the activity of CRISPR-Cas9 in genome engineering applications.