A mechanistic framework for DNA recognition and cleavage by Type V CRISPR-Cas effector nucleases

V 型 CRISPR-Cas 效应核酸酶 DNA 识别和切割的机制框架

基本信息

批准号：
BB/S001239/1
负责人：
Mark Dominik Szczelkun
金额：
$ 60.99万
依托单位：
University of Bristol
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2019
资助国家：
英国
起止时间：
2019 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FS001239%2F1
关键词：
mechanistic framework DNA recognition cleavage

项目摘要

It was demonstrated over 30 years ago that new genetic information could be inserted into the genomes of cultured human cells in the lab. This revolutionary discovery opened up the possibility of not only studying gene function but also correcting genetic mistakes that lead to disease. However, the process was inefficient, requiring millions of cells to be screened to find just one that had swapped a gene. It was realised that this process could be improved by introducing an enzyme into the cells that could break the DNA at the gene of interest (i.e. to cut both DNA strands using a so-called nuclease), and allowing the cell's natural DNA repair processes to do the rest. However, this required the development of "molecular scissors" that would cut just one gene amongst billions of other potential targets. Most research efforts concentrated on protein re-engineering of enzymes to recognise a user-defined sequence of DNA bases (and thus a unique gene). However, these tools were difficult to work with and the search continued for simpler programmable enzymes.A breakthrough came with the discovery in the early 2000s of bacterial enzyme systems that prevent viral infection, called Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) and CRISPR-associated (cas) genes. CRISPR-Cas systems had molecular scissors that recognised DNA in a unique way; a CRISPR nuclease separated the DNA strands and inserted an RNA molecule (called a "crRNA") to read out the DNA sequence, so producing a DNA-RNA hybrid (called an "R-loop"). CRISPR systems were easier to reprogram as only the RNA had to be changed and this was trivial for scientists compared to enzyme re-engineering. The CRISPR-led revolution in gene editing ignited in 2012 with the characterisation of CRISPR Cas9 and the first demonstrations of gene editing by Cas9 in human cell culture. A great deal of research has now been done using Cas9: it has been adapted for a wide range of genetic engineering functions, both in the lab and in the clinic; and we have a good understanding of how it works. Cas9 is fast becoming a common tool for basic, synthetic and clinical research.The Cas12a family of CRISPR nucleases have a similar biological function to Cas9 but were first characterised only in 2015. The structures of Cas12a enzymes are similar to Cas9 and they also appear to recognise gene sequences using crRNA-guided R-loops. However, there are key differences in the protein structures and we currently do not understand exactly how Cas12a works. It is important that we do so as it appears that Cas12a may be a better gene editing tool than Cas9. It has lower off-target cleavage in cells, meaning that the molecular scissors cut in the wrong place less often. Why this is the case is not known. The overall goal of this project is to establish more clearly how Cas12a forms an R-loop and cleaves the DNA, and how this is influenced by the crRNA and DNA sequences.To study Cas12a, we will use a combination of biochemistry and biophysics using purified proteins, DNA and RNA. Our principal technique is Magnetic Tweezers Microscopy. This "single-molecule" approach can observe R-loop formation by just one enzyme on one DNA molecule. We will seek to understand how the R-loop forms, how this is influenced by Cas12a, how changes in the crRNA affect the dynamics, and how incorrect pairing between the DNA and crRNA alter how the scissors cut the DNA. We will follow the DNA cleavage process and map where the cleavage occurs using a single-molecule DNA sequencing technique, called nanopore sequencing. And we will follow how different parts of the Cas12a protein move in relation to one another and to the DNA, by labelling with fluorescent markers. The culmination of these studies will be a fuller understanding of how Cas12a works and why it is more accurate. This will form the basis of future studies to improve Cas12a, and to further adapt it as the next generation of gene editing tools.

30年前，可以将新的遗传信息插入实验室中培养的人类细胞的基因组中。这一革命性发现不仅开辟了研究基因功能，而且还纠正导致疾病的遗传错误的可能性。但是，该过程效率低下，需要筛选数百万个细胞才能找到一个换了基因的细胞。人们意识到，可以通过将酶引入可以在感兴趣的基因上打破DNA的细胞（即使用所谓的核酸酶切割两个DNA链），并允许细胞的天然DNA修复过程到达细胞，从而改善此过程。剩下的。但是，这需要开发“分子剪刀”，这只会在其他数十亿个潜在靶标中切下一个基因。大多数研究工作都集中在酶的蛋白质重新设计上，以识别用户定义的DNA碱基序列（因此是独特的基因）。但是，这些工具很难使用，并且搜索继续以进行更简单的可编程酶。突破性的是2000年代初期的发现，可防止病毒感染的细菌酶系统，这些酶系统被称为聚集，散布，短，短裂（CRISPR）和短篇小说（CRISPR）和短篇小说（CRISPR）和短。 CRISPR相关（CAS）基因。 CRISPR-CAS系统的分子剪刀以独特的方式识别DNA。 CRISPR核酸酶分离了DNA链，并插入了RNA分子（称为“ CrRNA”）以读取DNA序列，因此产生DNA-RNA杂种（称为“ R-Loolop”）。 CRISPR系统更容易重新编程，因为与酶的重新设计相比，这对于科学家来说是微不足道的。 CRISPR领导的基因编辑革命于2012年以CRISPR CAS9的特征以及CAS9在人类细胞培养中编辑的第一次示范。现在，使用CAS9进行了大量研究：在实验室和诊所中，它已适用于广泛的基因工程功能；我们对它的工作原理有很好的了解。 CAS9迅速成为基本，合成和临床研究的常见工具。CAS12ACRISPR核酸酶家族具有与CAS9相似的生物学功能，但仅在2015年首次表征。CAS12A酶的结构与Cas9相似，它们似乎也似乎与Cas9相似。使用CRRNA引导的R环识别基因序列。但是，蛋白质结构存在关键差异，我们目前不了解CAS12A的工作原理。重要的是，我们这样做很重要，因为看来Cas12a可能是比Cas9更好的基因编辑工具。它在细胞中具有较低的脱靶裂解，这意味着分子剪刀在错误的位置切下的频率较低。为什么不知道这种情况。该项目的总体目标是更清楚地建立CAS12A如何形成R环并裂解DNA，以及它如何受到CRRNA和DNA序列的影响。为了研究Cas12a，我们将使用纯化的生物化学和生物物理学的组合使用纯化蛋白质，DNA和RNA。我们的主要技术是磁性镊子显微镜。这种“单分子”方法只能通过一个DNA分子上的一个酶观察到R环的形成。我们将寻求了解R-loop的形成，如何受到Cas12a的影响，CRRNA的变化如何影响动力学以及DNA和CRRNA之间的不正确配对如何改变剪刀切割DNA的方式。我们将遵循DNA裂解过程，并绘制使用单分子DNA测序技术（称为纳米孔测序）发生裂解的位置。我们将通过用荧光标记标记Cas12a蛋白的不同部分与彼此以及DNA的移动方式。这些研究的高潮将对CAS12A的工作原理以及为什么更准确。这将构成未来研究的基础，以改善CAS12A，并将其进一步适应下一代基因编辑工具。