Generation of genome-wide CRISPR knockout libraries for pig and chicken

在牲畜中进行选择性育种以保持和改善有益性状的益处在农业中早已实践。诸如体型增大、产奶量和抗感染性等性状在很大程度上是由猪、牛、羊和鸡等家畜动物的DNA编码中相对较小的差异决定的。最近的技术进步使科学家能够更详细、更快、更少地阅读动物的遗传密码。以至于我们开始了解动物基因组的特定变化如何导致特定的有益和有害特征。在不久的将来，目标是能够完全基于动物的DNA密码来预测和选择后代的有益特征。虽然我们阅读动物遗传密码的能力已经大大提高，但我们将遗传密码转化为物理属性的能力却远远落后。这是因为我们并不完全了解大多数基因的功能。了解特定基因功能的一个核心方法是删除该基因，并观察其对动物或动物细胞的影响。最近的进展使科学家能够独立地抑制人类细胞内的每一个基因，使他们能够询问特定的基因如何参与特定的生物过程。这些方法使我们能够将人类细胞的遗传密码与特定的生物特征或过程联系起来。这些技术还没有在牲畜物种中开发，由于所涉及的成本和相对缺乏与人类基因组相比的牲畜基因组的详细知识。在过去的几年中，一种新的技术已经开发出来，使科学家能够快速和特异性地编辑动物的基因组。这些方法已被证明是更有成本效益和技术上级优于以前的策略抑制基因。目前，该技术仅限于少数物种，如人类和小鼠。在本申请中，我们建议开发用于鸡和猪这两种最重要的家畜动物的这种技术。实现这一目标需要对这些动物的基因组序列的最新详细了解以及开发该技术所需的技术技能。在罗斯林研究所，我们有两名参与绘制猪和鸡基因组的主要研究人员，以及对新开发技术具有广泛知识的研究人员。此外，我们还将与最初开发该技术的美国布罗德研究所密切合作。因此，罗斯林研究所在成功开发和应用这项技术方面具有独特的优势。流感病毒是人类和家畜的重要病原体。禽流感病毒在禽类和猪等牲畜中的传播导致了禽流感病毒在人类中的爆发，包括潜在的大流行危险。在许多情况下，宿主（如人、鸡和猪）表达的基因是病毒所需的，可以限制病毒的复制。大量的工作致力于了解哪些基因在感染人类细胞后是重要的。在其他物种中，回答同样问题的研究相对较少。我们将使用这项技术来回答这些问题。虽然这将是研究的一个重要方面，但这只是这项新技术对从事畜牧动物研究的科学家有益的众多方式之一。这项技术有可能大大提高我们理解基因组中编码的基因之间的联系及其所起的功能作用的能力。这些进展将对改善牲畜育种的潜力产生重大影响，也将有助于我们对生物学的基本理解。

The benefits of selective breeding within livestock to maintain and improve beneficial traits have long been practiced in agriculture. Traits such as increased size, milk production and resistance to infection are, for the most part, determined by relatively small differences in the DNA code of livestock animals such as pigs, cows, sheep and chickens. Recent technological advances allow scientists to read the genetic code of animals in greater detail, faster and for less money. So much so that we are beginning to understand how specific changes in the genomes of animals result in specific beneficial and detrimental characteristics. The aim in the near future is to be able to predict and select for beneficial characteristics in offspring based entirely on the animals DNA code. Although our ability to read the genetic code of animals has greatly improved our ability to translate the genetic code into physical attributes lags far behind. This is because we don't fully understand the function of most genes. A central approach to understanding the function of a particular gene involves deleting the gene and observing the effect this has on the animal or the animal's cells. Recent advances have allowed scientists to independently inhibit every individual gene within human cells, allowing them to ask how particular genes are involved in specific biological processes. These approaches allow us to link the genetic code of human cells to particular biological traits or processes. Such technologies have not been developed in livestock species due to the cost involved and comparative lack of detailed knowledge of livestock genomes compared to human genomes.In the last few years a new technology has been developed that allows scientists to rapidly and specifically edit the genomes of animals. These approaches have been shown to be more cost effective and technically superior to previous strategies for inhibiting genes. Currently the technique is limited to only a few species such as human and mouse. In this application we propose developing this technology for chicken and pig two of the most important livestock animals. Achieving this requires the latest detailed knowledge of the genome sequence of these animals and the technical skills required to develop the technology. At the Roslin institute we have two of the leading researchers involved in mapping the pig and chicken genomes as well as researchers with extensive knowledge of the newly developed technology. In addition we will be working closely with the Broad institute in the US who originally developed the technology in question. Therefore the Roslin Institute is uniquely positioned to successfully develop and apply this technology.Initially we will be asking a relatively straightforward question using the technology. Influenza virus is an important pathogen both in humans and in livestock animals. Spread of the virus in livestock such as birds and pigs contributes to outbreaks of the virus in humans, including potentially dangers pandemics. In many cases genes expressed by the host (such as human, chickens and pigs) are required by the virus and can restrict the replication of the virus. Significant amounts of work has been dedicated to understanding which genes are important following infection of human cells. Relatively little research has been performed to answer the same question in other species. We will use this technology to answer these questions. Although this will be an important aspect of the research, it is only one of many ways in which this new technology will be of benefit to scientists working on livestock animals. This technology has the potential to greatly improve our ability to understand the link between the genes encoded in a genome the functional roles they play. Such advances will have major impacts on the potential for improved breeding of livestock and will also contribute to our basic understanding of biology.