Cell：CRISPR-Cas的生物學

CRISPR-Cas的生物學：向後和向前

摘 要

在細菌和古細菌中，成簇規則間隔短迴文重複序列（CRISPR）和CRISPR相關（Cas）蛋白構成針對噬菌體和其他外源遺傳元件的適應性免疫系統。在這裡，我們回顧了多種CRISPR-Cas系統的生物學特徵，以及近年來在理解CRISPR-Cas免疫的三個階段（適應，crRNA生物發生和干擾）的潛在機制方面取得的重大進展。還討論了CRISPR-Cas在噬菌體感染背景下的生態學和調控，這些系統在免疫領域的作用以及推動該領域前進的開放性問題。

In bacteria and archaea, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system against phages and other foreign genetic elements. Here, we review the biology of the diverse CRISPR-Cas systems and the major progress achieved in recent years in understanding the underlying mechanisms of the three stages of CRISPR-Cas immunity: adaptation, crRNA biogenesis, and interference. The ecology and regulation of CRISPR-Cas in the context of phage infection, the roles of these systems beyond immunity, and the open questions that propel the field forward are also discussed.

引言:超過十億年的共同進化已經在原核生物和它們的病毒捕食者中產生了多種複雜的逃避和防禦策略。噬菌體吸附的抑制，入侵噬菌體基因組的限制性修飾以及流產感染是針對噬菌體捕食的充分特徵的先天防禦機制之一（Labrie等，2010）。過去幾十年微生物學中最令人興奮的發現之一是原核生物也顯示適應性，遺傳免疫性的啟示。原核CRISPRCas適應性免疫系統存儲過去感染的記憶，並且在再感染後，部署用於序列特異性沉默噬菌體和其他移動遺傳元件（MGE）（例如質粒和轉座子）的RNA引導的核酸酶。

INTRODUCTION:More than a billion years of coevolution have produced a multitude of sophisticated evasion and defense strategies in prokaryotes and their viral predators. Suppression of bacteriophage adsorption, restriction modification of the invading phage genome, and abortive infection are among the well-characterized innate defense mechanisms against phage predation (Labrie et al., 2010). One of the most exciting discoveries in microbiology of the last decades was the revelation that prokaryotes also display adaptive, heritable immunity. Prokaryotic CRISPRCas adaptive immune systems store memory of past infections, and upon reinfection, deploy RNA-guided nucleases for sequence-specific silencing of phages and other mobile genetic elements (MGEs), such as plasmids and transposons.

這些系統的特徵分別在完整的細菌和古細菌基因組的50％和近90％中發現（Grissa等，2007; Makarova等，2015），是CRISPR陣列。該基因組座位由交替的相同重複序列和獨特的間隔子組成（Ishino等，1987; Jansen等，2002）。 CRISPR研究的一個里程碑是認識到間隔序列與質粒和噬菌體基因組相匹配，因為它是CRISPR-Cas可能作為原核生物防禦機制的第一個暗示（Bolotin et al。，2005; Mojica et al。，2005; Pourcel等，2005）。 CRISPR-Cas作為適應性免疫系統的功能，其中CRISPR陣列用作先前感染的檔案，最終通過觀察到嗜熱鏈球菌的噬菌體挑戰通過獲得免疫的噬菌體衍生間隔物來刺激CRISPR陣列的擴增抵抗隨後的感染（Barrangou等，2007）。通過顯示CRISPR驅動的表皮葡萄球菌中質粒結合和轉化的抑制（Marraffini和Sontheimer，2008），不久之後證明了免疫系統預防其他MGE感染的能力。

The defining feature of these systems, which are found in 50% and nearly 90% of complete bacterial and archaeal genomes, respectively (Grissa et al., 2007; Makarova et al., 2015), is the CRISPR array. This genomic locus is composed of alternating identical repeats and unique spacers (Ishino et al., 1987; Jansen et al., 2002). A milestone in CRISPR research was the realization that the spacer sequences match plasmids and phage genomes, as it was the first hint that CRISPR-Cas might function as a prokaryotic defense mechanism (Bolotin et al., 2005; Mojica et al., 2005; Pourcel et al., 2005). The function of CRISPR-Cas as an adaptive immune system in which the CRISPR array serves as an archive of previous infections was ultimately demonstrated by the observation that phage challenge of Streptococcus thermophilus stimulates expansion of the CRISPR array by acquisition of phage-derived spacers that immunize against subsequent infection (Barrangou et al., 2007). The ability of the immune system to prevent infection by other MGEs was demonstrated shortly afterward by showing CRISPR-driven inhibition of plasmid conjugation and transformation in Staphylococcus epidermidis (Marraffini and Sontheimer, 2008).

與CRISPR陣列相鄰的是一系列編碼Cas蛋白的基因，其驅動免疫的三個階段：適應，CRISPR RNA（crRNA）生物發生和干擾。在適應期間，選擇，處理外來核酸並整合到CRISPR陣列中以提供感染記憶。 當CRISPR陣列被轉錄以產生在重複序列內處理以產生成熟crRNA的長前體crRNA（pre-crRNA）時，檢索到記憶。 在隨後的感染中，干擾機制由crRNAs引導以切割外源核酸中稱為原始空間的互補序列（圖1）。通過折衷由MGE編碼的自私的，往往是敵對的程序，CRISPRCas系統保護原核生物免於感染。 根據cas基因的分類和干擾複合物的性質，CRISPR-Cas系統被分為兩類，它們被進一步細分為六種類型和幾種亞型，每種亞型都具有特徵性的cas基因。 1類CRISPR-Cas系統（類型I，III和IV）採用多Cas蛋白複合物進行干擾，而在2類系統（II，V和VI類）中，干擾通過單一效應蛋白實現（Makarova et 2011,2013,2015; Shmakov等，2015）。

Adjacent to the CRISPR array is a series of genes encoding the Cas proteins that drive the three phases of immunity: adaptation, CRISPR RNA (crRNA) biogenesis, and interference. During adaptation, foreign nucleic acids are selected, processed, and integrated into the CRISPR array to provide a memory of infection. Memory is retrieved when the CRISPR array is transcribed to produce a long precursor crRNA (pre-crRNA) that is processed within the repeat sequences to yield mature crRNAs. Upon subsequent infection, the interference machinery is guided by crRNAs to cleave complementary sequences, termed protospacers, in the foreign nucleic acids (Figure 1). By compromising the selfish, often hostile programs encoded by MGEs, CRISPRCas systems protect prokaryotes from succumbing to infection. According to the assortment of cas genes and the nature of the interference complex, CRISPR-Cas systems have been assigned to two classes, which are further subdivided into six types and several subtypes that each possess signature cas genes. Class 1 CRISPR-Cas systems (types I, III, and IV) employ multi-Cas protein complexes for interference, whereas in class 2 systems (types II, V, and VI), interference is accomplished by a single effector protein (Makarova et al., 2011, 2013, 2015; Shmakov et al., 2015).

喜歡這篇文章嗎？立刻分享出去讓更多人知道吧！