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As indicated above, RNAi plays an essential role in many plant biological processes, including developmental timing and patterning, transposon control, DNA methylation, and chromatin modification. Although its role in human immunity is not known yet, RNAi constitutes the primary natural plant immune system against viruses. The plant RNAi defense system is highly specific because it will only target nucleic acids that are identical in sequence to the triggering dsRNAs. In addition to viral infections, RNAi protects the genome from genetics parasites such as transposons and retrotransposons. These mobile nucleic acid elements can cause diseases and alter the genome in a number of different ways.
RNAi 在植物生长发育过程中扮演着关键角色，例如调控发育时机和形态、控制转座子活性、调控 DNA 甲基化水平以及染色质修饰等。尽管目前尚未阐明 RNAi 在人体免疫系统中的作用，但它是植物抵御病毒侵害的主要天然防御手段。植物 RNAi 防御系统具有很高的特异性，只会靶向与触发性双链 RNA (dsRNA) 完全匹配的核酸序列。除了防御病毒感染，RNAi 还可保护植物基因组免受转座子和反转录转座子等移动核酸元件的侵害。这些游走基因组的元件可能会导致疾病并通过多种途径改变植物基因组。
By analogy to RNAi-mediated plant protection against pathogen attack, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) loci and CRISPR-associated (Cas) proteins provide acquired immunity against viruses and other mobile genetic elements in bacteria and archaea. These CRISPR sequences are part of the bacteria’s immune system and provide endogenous adaptive immunity in approximately 40% of bacterial genomes and 70% of sequenced archaeal species. Immunological memories of phages and plasmids are stored in the CRISPR array as short spacer sequences that intercalate between repeats and specify the targets of CRISPR-Cas immunity. The locus consists of CRISPR-associated (Cas) genes in operons in addition to the spacer-repeat array. These repeats were initially discovered in the 1980s in E. coli, but their function was not uncovered until 2007 by Barrangou and colleagues, who demonstrated that the Streptococcus thermophilus strains that survived initial phage challenge were resistant to subsequent phage challenge and had incorporated sequences from the phage genome into their CRISPR arrays. When a new infection occurs, the spacer-repeat array is expressed as a long precursor pre-CRISPR RNA (pre-crRNA), and the transcript is processed into individual spacer repeat units as crRNAs, while the cas genes are transcribed and translated into proteins. Additionally, the Cas genes are transcribed into proteins. Such crRNAs associate with and direct Cas nucleases (e.g., Cas9) to cleave foreign nucleic acids complementary to the spacer sequence. Cleavage of the viral or plasmid target DNA prevents infection (Fig. 4). Target recognition requires base pairing between the crRNA and the target and a PAM sequence adjacent to the crRNA-binding region. The requirement of a PAM site prevents cleavage of the CRISPR loci, which lacks a PAM site. The PAM sequences allow the discrimination between self and non-self, a crucial characteristic of all immune systems.
类似于 RNAi 介导的植物对抗病原体攻击的情况，Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) 序列和 CRISPR 相关（Cas）蛋白为细菌和古细菌提供了对抗病毒和其他可移动遗传因子的获得性免疫。这些 CRISPR 序列是细菌免疫系统的一部分，在约 40% 的细菌基因组和 70% 的已测序古菌物种中发挥着内源性适应性免疫功能。细菌可以将曾经感染过的噬菌体和质粒片段 (间隔序列) 插入 CRISPR 阵列作为“免疫记忆”，指导 Cas 蛋白识别并切割外来核酸。CRISPR 阵列除了包含间隔序列，还包含多个间隔序列之间的一段重复序列以及多个编码 Cas 蛋白的基因。CRISPR 系统的功能于 20 世纪 80 年代在大肠杆菌中被发现，但直到 2007 年科学家才阐明了其详细机制。科学家们发现能够抵抗噬菌体侵染的嗜热链球菌株，其 CRISPR 阵列整合了来自噬菌体基因组的片段。当细菌遭遇新的病毒或质粒侵染时，CRISPR 阵列会首先转录成前体前 CRISPR RNA (pre-crRNA)，然后被加工成一个个小的 crRNA 分子，同时 Cas 基因也会转录成 Cas 蛋白。crRNA 分子可以引导 Cas 核酸酶 (例如 Cas9) 切断与 crRNA 序列互补的外来核酸 (例如病毒或质粒 DNA)，从而起到清除入侵者的作用 (见图 4)。CRISPR 系统识别靶标需要 crRNA 和靶标序列以及靠近 crRNA 结合区域的 PAM 序列配对。PAM 序列的存在可以防止 Cas 蛋白切割细菌自身的 CRISPR 阵列 (因为 CRISPR 阵列本身不包含 PAM 序列)，从而区分“自己”和“非自己”，这是免疫系统的重要特性。
Fig. 4 Schematic representation of CRISPR-Cas systems. When a phage infects a bacterium, it injects its DNA into the bacterium which responds by producing Cas enzymes to cut the phage DNA into small fragments and then incorporate them into its own genome at the CRISPR locus. During the second infection, the bacterium that survived the first infection activates the CRISPR locus to generate small RNA molecules and Cas protein enzymes. After RNA processing, each small RNA molecule associates with the Cas effector protein and guides the enzyme to cleave the invading phage DNA, thus blocking the infection. Cas genes code for important endonuclease enzymes in this pathway One of these, Cas9, is a nuclease that cuts nucleic acid (DNA or RNA).CRISPR-Cas 系统的示意图。当噬菌体感染细菌时，它会将自己的 DNA 注入细菌细胞。作为回应，细菌会产生 Cas 蛋白酶，将噬菌体 DNA 切割成小片段，然后将其整合到自身基因组的 CRISPR 区域。在第二次感染过程中，存活下来的细菌会激活 CRISPR 区域以产生小 RNA 分子和 Cas 蛋白酶。经过 RNA 加工后，每个小 RNA 分子都会与 Cas 执行蛋白结合，并引导 Cas 蛋白酶切割入侵的噬菌体 DNA，从而阻止感染。Cas 基因在这个过程中编码重要的核酸内切酶。其中之一，Cas9 是一种核酸酶，可以切割核酸 (DNA 或 RNA)。
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CRISPR/Cas9 genome editing is a relatively new nucleic acid-based technique that relies on high-performing nucleic acids to produce highly efficient and specific alterations to the genome. The technology has been adapted for genome editing applications in eukaryotic cells and is an emerging tool because it allows for some more flexibility compared to other gene-editing tools. Moreover, the technique is incredibly precise, cheap, and easy. The CRISPR-Cas system described so far falls into two major classes, which are further divided into a total of six types. Class 1 uses a multi-subunit effector complex and consists of CRISPR type I, II, and IV. Class 2 employs a single effector nuclease and consists of CRISPR type II, V, and VI. CRISPR type II is known by Cas9 nuclease, a multidomain protein that is able to bind crRNA and cleave corresponding target DNA and thus represents the most convenient format to use in eukaryotic cells. Indeed, in a landmark 2012 paper, Doudna, Chapentier, and Jinek showed that they could use CRISPR-Cas 9 system to introduce specific mutations in any genome. When loaded with both crRNA and tracrRNA (or an artificial chimera of the two), the wild-type Cas9 can site-specifically introduce double-stranded DNA breaks (DSBs). The resulting DSB will either generate nonspecific mutations knocking out a gene through the error-prone NHEJ (non-homologous end joining) pathway. Alternatively, if a donor template with homology to the target locus is supplied, the DSB may be repaired by the homology-directed repair (HDR) pathway allowing for precise replacement mutations to be made. By directing the cells toward one of these repair mechanisms, CRISPR/Cas9 can be used to generate knockouts or knock-ins in cells. In addition to gene editing, the technology was also adapted for mRNA targeting (knockdown) with high specificity. This system relies on an enzyme called Cas13a, which complexes with guide RNA, which binds to complementary messenger RNA. The better the match between the guide RNA and the messenger RNA, the more effective the Cas13a’s gene silencing action.
CRISPR/Cas9 基因编辑是一种相对较新的基于核酸的技术，它利用高效的核酸工具实现对基因组进行高效特异的修饰。这项技术因其相较于其他基因编辑工具更灵活而被改用于真核细胞的基因编辑领域，并且以其惊人的精确性、低廉的成本和简便的操作流程成为新兴工具。迄今为止描述的 CRISPR-Cas 系统可以分为两大类，这两大类又可进一步细分为六种类型。第一类使用多亚基效应复合体，包含 I、II 和 IV 型 CRISPR 系统。第二类使用单个效应核酸酶，包含 II、V 和 VI 型 CRISPR 系统。CRISPR II 型以 Cas9 核酸酶著称，Cas9 是一种能够结合 crRNA 并切割对应靶标 DNA 的多域蛋白，因此它是真核细胞中最方便使用的格式。2012 年的一篇里程碑式论文中，Doudna, Chapentier 和 Jinek 证明了他们可以使用 CRISPR-Cas9 系统在任何基因组中引入特定突变。当 Cas9 同时加载 crRNA 和 tracrRNA (或两者的人工融合体) 时，野生型 Cas9 可以定位特异地引入双链 DNA 断裂 (DSB)。产生的 DSB 要么会通过容易出错的非同源末端连接 (NHEJ) 途径产生非特异性突变，从而敲除基因；或者，如果提供具有靶向位点同源性的供体模板，则 DSB 可以通过同源定向修复 (HDR) 途径进行修复，从而实现精确的替换突变。通过将细胞导向这些修复机制之一，CRISPR/Cas9 可以用于在细胞中产生敲除或敲入。除了基因编辑之外，该技术还被用于靶向 mRNA (敲减) 并实现高特异性。该系统依赖于一种名为 Cas13a 的酶，Cas13a 与引导 RNA 结合，后者与互补的信使 RNA 结合。引导 RNA 与信使 RNA 的匹配程度越好，Cas13a 的基因沉默作用就越有效。
In contrast to previous tools for genome editing such as zinc-finger nucleases and transcription activator-like effector nucleases, CRISPR does not require engineering of new enzymes for each new target. With respect to specificity, high-fidelity Cas9 variants including eSpCas9, SpCas9-HF1, HypaCas9, and xCas9 were developed. These Cas9 protein variants combined with the use of chemically modified gRNAs have increased the specificity of genome editing. Although RNAi was adopted as gene silencing technique first, CRISPR has surpassed RNAi in popularity due to several advantages including specificity and versatility. Drs Emmanuelle Chapentier and Jennifer Doudna who developed the CRISPR technology are awarded the Nobel Prize in Chemistry 2020.
与锌指核酸酶和转录激活因子样效应物核酸酶等以往的基因组编辑工具相比，CRISPR 技术不需要针对每个新靶点设计新的酶。在特异性方面，科学家们开发了高效的 Cas9 变体，例如 eSpCas9、SpCas9-HF1、HypaCas9 和 xCas9。这些 Cas9 蛋白变体结合化学修饰的 gRNA 使用，提高了基因组编辑的特异性。尽管 RNAi 技术较早被用于基因沉默，但由于特异性和通用性等诸多优势，CRISPR 技术已经超越了 RNAi 的流行程度。CRISPR 技术的开发者 Emmanuelle Chapentier 博士和 Jennifer Doudna 女士也因此共同获得了 2020 年诺贝尔化学奖。
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Analysis of RNAi in a variety of organisms revealed that RNAi is a widespread natural phenomenon that is conserved across fungi, plants, and animals. The process is induced not only by exogenous siRNAs but also by several endogenous small RNA species such as microRNAs and piwi-interacting RNAs. While miRNAs participate actively in all cellular processes, piwi-interacting RNAs (piRNAs) have a major function in the repression of transposable elements in the germline. Recently, CRISPR/Cas9 technology has evolved as the most powerful approach to generate genetic models both for fundamental and preclinical research. Not only can scientists use such technology to silence genes by knocking them out, they can also introduce desired genes into the genome. The choice remains with the user to perform knockout, knock-ins, or knockdown experiments, making CRISPR interference extremely versatile.
RNAi 技术在不同生物体中的分析揭示了 RNAi 是一种广泛存在的自然现象，在真菌、植物和动物中都保守存在。RNAi 不仅可以由外源性 siRNA 引发，还可以由几种内源性小 RNA 分子（例如 microRNA 和 piRNA）引发。microRNA 积极参与所有细胞过程，而 piRNA 则主要在生殖细胞中抑制转座子元素。CRISPR/Cas9 技术最近已发展成为产生用于基础和临床前研究的基因模型的最强大方法。科学家们不仅可以使用这种技术通过敲除的方式实现基因沉默，还可以将所需的基因引入基因组。CRISPR 干扰技术使用非常灵活，用户可以选择进行敲除、敲入或敲减实验。
