编者按：4月21日，EASL2017聚焦基础科学研究。“Basic science highlights: CRISPR-Cas: from discovery to applications”专题会议上，西班牙Lluis MONTOLIU教授和法国Jean-Paul CONCORDET教授分别介绍了对CRISPR-Cas系统的探索和CRISPR-Cas系统在基因操作中的应用。会后，本刊特邀两位教授为我们介绍CRISPR-Cas机制，及其如何为医学研究服务、为疾病治疗谋福利。

 

CRISPR-Cas介导的防御过程分为几个主要步骤？

 

Montoliu教授指出CRISPR-Cas是一种非常古老的防御机制，其在细菌的原核世界中非常有效，而且在数十亿年前就已经开始这样工作了。当病毒感染细菌时，病毒将自己的基因组导入细菌体内，一旦产生基因组，基因组将会被消化，而其中的一些片段也将会被整合到细菌的基因组中。通过这种方式，细菌将获得抵抗相同病毒再次感染的能力。这是因为病毒基因组整合到细菌中的片段已经在转录，在这过程中，短的RNA分子在细菌体内不断循环。当细菌再次被同一病毒感染时，因为它们具有同一序列，病毒DNA将与这些短RNA相匹配并结合，最终被细菌消化。所以，它是一种在细菌分子水平上运行的非常有效的获得性的防御系统。这种“后天性”获得的防御，也会传递到后代的细菌中。

 

Concordet教授还谈到如果查阅细菌的基因组，可以了解其暴露于不同的病原体（噬菌体和质粒）的情况。仔细看这小部分居住在基因组的部分，它可提供了一个洞察该细菌菌株的历史。这种机制有许多应用。例如，在乳品工业中，为了生产奶酪或酸奶，必须生产大量的细菌，生产者不希望细菌被感染。利用该机制，生产者可有效地对其菌株进行疫苗接种，并选择使用对可能暴露病原体具有抵抗力的那些菌株。因此，CRISPR-Cas不仅在基因组编辑，在微生物学上同样也具有潜在的巨大影响。

 

Prof. Montoliu: This is a very ancient mechanism, which has proven to be very effective in the prokaryotic world of bacteria, and they have been doing it for literally billions of years. When a virus infects a bacterium, they introduce their genome into the bacteria and as soon as they produce the genome, the genome is going to be digested and some of these pieces are going to be integrated into the genome of the bacteria. By doing that, the bacteria become resistant to being infected again by the same viruses. This occurs because the pieces of viral genome that are integrated into the bacteria have transcribed and in the process, the short RNA molecules are constantly circulating inside the bacteria. The next time the same virus visits the bacteria, these short RNAs will match with the viral DNA because they are in the same sequence, and be bound, ultimately being digested by the bacteria. So it is a very effective acquired system of defense. It is an immune system that operates at a molecular level in the bacteria. It is a “learned” defense that is transferred to the progeny of the bacteria.

 

Prof. Concordet: It is also interesting that if you go through the genome of the bacteria, you can get an idea of the different pathogens it has been exposed to (phages and plasmids). You can look at the part of the genome where these small pieces are residing and it provides an insight into the history of that bacterial strain. There are many applications for this mechanism. For example, in the dairy industry, where it is necessary to grow large volumes of bacteria for cheese or yoghurt production, they do not want their bacteria to be infected. They can effectively vaccinate their bacterial strains and select those that have acquired resistance to pathogens they might be exposed to. There is a potentially huge impact in microbiology as well, not only in genome editing.

 

除免疫作用外，CRISPR-Cas还参与哪些过程？

 

细菌中，CRISPR-Cas系统真正的生物学功能是专门针对病原体和外来DNA进行免疫和防御。在原核世界中很少会形成发展这样用于一个目的机制。通常，这种机制将用于各种不同的目的。Montoliu教授在采访中介绍到，一些微生物研究表明，除了作为原核免疫基础机制之一，CRISPR-Cas系统也可调控基因的表达。显然，有些基因可以通过CRISPR-Cas进行调控，所以这种协同进化的免疫机制已经适应了调控基因。几个月前，有研究报道，有一种细菌具有该核酸酶的抑制剂。这种细菌产生抑制该Cas9核酸酶的产物，使它不会自动消化其自身的基因组。在这种特殊的细菌中，噬菌体已经插入到细菌基因组中，所以它能够存活下来。此外CRISPR-Cas还有其他方面的应用。据报道，已经发现有病毒从细菌中劫持了CRISPR-Cas系统，并且开发了自己的CRISPR-Cas系统，用于对抗细菌宿主的CRISPR-Cas免疫系统，这些机制作用始终在进化当中。

 

Prof. Concordet: The real biological functions of the CRISPR-Cas system in bacteria are specifically aimed at immunity and defense against pathogens and foreign DNA.

 

Prof. Montoliu: Very rarely in nature in the prokaryotic world would such a mechanism develop and then be used for a single purpose. Normally, such a mechanism would be used for a variety of different purposes. Some microbiological studies suggest that besides being the basis of one of the prokaryotic immune mechanisms, the CRISPR-Cas system can also be used for gene regulation. Apparently, there are certain genes that can be regulated through CRISPR-Cas, so there is a coevolution of this immune mechanism that has adapted to regulate genes. Very recently, just a couple of months ago, it was reported that there is a bacteria which has the first inhibitor for this nuclease. This bacterium produces a product that inhibits this Cas9 nuclease so that it won’t autodigest its own genome. In this particular bacteria, the phage is already inserted in the bacterial genome, so it is able to survive. I assume there are other ways in which CRISPR-Cas is being used. It has been reported that viruses have kidnapped CRISPR-Cas systems from bacteria and have themselves developed CRISPR-Cas against the host CRISPR-Cas - an immune system from the virus against the immune system of the bacteria. These mechanisms are evolving all the time.

 

CRISPR-Cas在医学科学中有哪些应用？

 

Concordet教授指出，CRISPR-CAS在基础研究的主要应用是在进一步推进知识和基础科学进步，同时也有助于更好地了解疾病和基因致病的过程。这个最重要的应用已经在世界各地的多数实验室中进行，中国也做出了非常重要的贡献。在治疗和潜在的治疗方面，其有可能是修改基因和修正导致疾病的基因突变的重要手段。我们非常希望利用CRISPR-Cas进行基因组编辑，这将使我们能够修复一些疾病相关的突变。但在具体操作时也会遇到许多限制和挑战，如试剂的传递和任何影响整体效率和程度的临床因素。所有这些因素将被深入研究。我们希望基于CRISPR-Cas的基因治疗可以对一些疾病的治疗做出重要贡献。

 

Montoliu教授谈到，CRISPR-Cas在医学领域主要有两个潜在的应用。除基础研究这个根本外，CRISPR-Cas还可以以更加个性化的方式介入疾病中。我们可以根据特定患者发生的突变来模拟疾病，而不是像近三十多年来我们在遗传学上进行的，创造出一种疾病的细胞或动物模型，这很容易产生突变。目前已经可以完全重现特定疾病患者发生的突变。在医学上，有时说没有疾病，只有患者。将来我们不仅有可能重现一个遗传疾病的动物模型，还可以是一个给定的人类个体的动物模型。通过研究这些特定人类个体模型，我们将更好地了解疾病的起源过程进而有助于开发相应的治疗方法。同时希望这种技术将是基因治疗的重要手段。

 

但当其进入临床应用之前，我们需要解决与使用CRISPR-Cas技术相关的限制和存在的问题。CRISPR-Cas对于我们正在进行的研究应用是非常好的，但仍然不能按我们的意愿精确控制产生等位基因，当我们生产出所需的等位基因时，我们也将生产许多其他等位基因。在处理细胞或动物时这很容易控制，但当应用到人身上时，这要难得多，所以需要仔细谨慎然后再进行。

 

Prof. Concordet: The main application of CRISPR-Cas is in basic research in furthering knowledge and basic science, and contributing also to medical science through a better understanding of disease and the genetics underpinning disease processes. This most important application is already taking place in most labs around the world, including in China where there are very important contributions being made. In terms of treatment and potential therapeutics, the concept might be to modify genes and the mutations that are responsible for disease. Of course, we very much hope that genome editing using CRISPR-Cas will allow us to repair some of the mutations responsible for disease. There are hopes, but there will be lots of limitations and challenges in implementing these approaches, such as the delivery of the reagents and the overall efficiency and extent of any clinical impact. All of these factors will be intensely studied. In some diseases, there is hope that gene therapy based on CRISPR-Cas can provide an important contribution.

 

Prof. Montoliu: CRISPR-Cas has two main potential applications in medicine. Basic research is fundamental. But CRISPR-Cas can also allow an approach to disease in a more personalized manner. We would be able to model a disease according to the mutations that occurs in a particular patient, rather than proceeding genetically and creating cellular or animal models of a given disease with easy to produce mutations as we have been doing for over thirty years. Now we can move to reproduce exactly the same mutations that have been diagnosed in a human patient. In medicine, it is said that there are no diseases, but only patients. Potentially, we could not only reproduce an animal model of a genetic disease, but also an animal model of a given human individual. By studying several of these human individual models, we will come to better understand the origins of the disease process that will help develop treatments. Later on, we envisage that this technology will be important in gene therapy approaches. But before things come into clinical use, we need to sort out the limitations and problems associated with the use of CRISPR-Cas technology. CRISPR-Cas is great for what we are using it for, but we still cannot precisely control the allele that we are willing to produce. While we produce the desired allele, we will also be producing many other alleles. This is easy to control when dealing with cells or animals, but when applied to people, it is much more difficult and we need to be prudent before proceeding there.