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

30多年前，科学家们证明了新的遗传信息可以插入到实验室培养的人类细胞的基因组中。这一革命性的发现不仅为研究基因功能开辟了可能性，而且也为纠正导致疾病的遗传错误开辟了可能性。然而，这个过程效率低下，需要筛选数百万个细胞才能找到一个交换了基因的细胞。人们意识到，这个过程可以通过向细胞中引入一种酶来改善，这种酶可以在感兴趣的基因处破坏DNA（即使用所谓的核酸酶切割两条DNA链），并允许细胞的天然DNA修复过程完成其余的工作。然而，这需要开发“分子剪刀”，在数十亿个其他潜在目标中仅切割一个基因。大多数研究工作集中在酶的蛋白质重组上，以识别用户定义的DNA碱基序列（从而识别独特的基因）。然而，这些工具很难使用，研究人员仍在继续寻找更简单的可编程酶。21世纪初，人们发现了一种预防病毒感染的细菌酶系统，称为规则间隔短回文重复序列（CRISPR）和CRISPR相关（cas）基因。CRISPR-Cas系统具有以独特方式识别DNA的分子剪刀; CRISPR核酸酶分离DNA链并插入RNA分子（称为“crRNA”）以读出DNA序列，从而产生DNA-RNA杂合体（称为“R-loop”）。CRISPR系统更容易重新编程，因为只需改变RNA，与酶重新工程相比，这对科学家来说微不足道。2012年，CRISPR领导的基因编辑革命点燃了CRISPR Cas9的特性，并在人类细胞培养中首次展示了Cas9的基因编辑。现在已经使用Cas9进行了大量的研究：它已经在实验室和临床上适应了广泛的基因工程功能;我们对它的工作原理有了很好的了解。Cas9正迅速成为基础、合成和临床研究的常用工具。Cas 12 a家族的CRISPR核酸酶具有与Cas9相似的生物学功能，但仅在2015年才首次被表征。Cas 12 a酶的结构与Cas9相似，它们似乎也使用crRNA引导的R环识别基因序列。然而，蛋白质结构存在关键差异，我们目前还不清楚Cas 12 a是如何工作的。我们这样做很重要，因为Cas 12 a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切割，这意味着分子剪刀在错误的地方切割的频率较低。为什么会出现这种情况尚不清楚。该项目的总体目标是更清楚地确定Cas 12 a如何形成R环并切割DNA，以及crRNA和DNA序列如何影响Cas 12 a。为了研究Cas 12 a，我们将使用纯化的蛋白质，DNA和RNA，使用生物化学和生物物理学相结合的方法。我们的主要技术是磁镊显微镜。这种“单分子”方法可以观察到一个DNA分子上的一种酶形成的R环。我们将试图了解R环是如何形成的，这是如何受到Cas 12 a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切割过程，并使用单分子DNA测序技术（称为纳米孔测序）绘制切割发生的位置。我们将通过荧光标记来跟踪Cas 12 a蛋白质的不同部分如何相互移动以及与DNA的关系。这些研究的最终结果将是更全面地了解Cas 12 a是如何工作的，以及为什么它更准确。这将成为未来研究的基础，以改善Cas 12 a，并进一步将其作为下一代基因编辑工具。

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.