CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is the microbial world’s answer to adaptive immunity. Like us, bacteria produce antibodies when they are invaded by a pathogen and do not produce antibodies in case they encounter the same pathogen again. Instead, they incorporate part of the pathogen’s DNA into their own genome and attach it to an enzyme. The enzyme uses it to recognize the pathogen’s DNA sequence and cut it if the pathogen reappears.
The enzyme that performs the cutting is called Cas, which stands for CRISPR Associated. Although the CRISPR-Cas system evolved as a bacterial defense mechanism, it has been utilized and adapted by researchers as a powerful tool for genetic manipulation in laboratory studies. Agricultural applications have also been demonstrated, and the first of his CRISPR-based treatments has just been approved in the UK for the treatment of sickle cell disease and transfusion-dependent beta-thalassemia.
Now, researchers have developed a new method to search the genome for CRISPR-Cas-like systems. And it turns out there may be a lot more tools we can use.
modification of DNA
To date, six CRISPR-Cas systems have been identified in various microorganisms. Although they differ in detail, they all have the same charm. This means delivering proteins to specific sequences of genetic material with a degree of specificity that has traditionally been technically difficult, expensive, and time-consuming to achieve. DNA sequences of interest can be programmed into the system and targeted.
Native systems found in microorganisms typically introduce nucleases, DNA-cutting enzymes, into sequences to chop up the pathogen’s genetic material. This ability to cut any DNA sequence of your choice can be used for gene editing. It can be used in conjunction with other enzymes and/or DNA sequences to insert or delete additional short sequences and correct mutated genes. Some CRISPR-Cas systems cut specific RNA molecules rather than DNA. These can be used to remove pathogenic RNA, similar to how it is removed in native bacteria, as well as the genomes of some viruses. It can also be used to rescue defects in RNA processing.
However, there are many other useful ways to modify nucleic acids. And it is an open question whether enzymes that perform additional modifications have evolved. So some researchers decided to look for them.
MIT researchers have developed a new tool to detect variable CRISPR arrays and applied it to 8.8 tera (1012) base pairs of prokaryotic genome information. Many of the systems they discovered are rare and have only appeared in datasets in the past decade, highlighting how important it is to continue adding previously hard-to-obtain environmental samples to these data repositories. I’m emphasizing.
The new tools are needed because protein and nucleic acid sequence databases are expanding at tremendous rates, and the technology for analyzing all that data must keep up. Some algorithms used to analyze them try to compare every sequence to every other sequence, but this is clearly unsustainable when dealing with billions of genes. Some rely on clustering, but these only detect very similar genes and cannot actually reveal evolutionary relationships between distantly related proteins. However, fast hashtag-based clustering with an emphasis on locality (“flash clustering”) splits billions of proteins into a small number of larger sequence clusters that differ slightly, allowing new and rare relatives to be identified. It works by identifying.
The search using FLSHclust successfully elicited 188 new CRISPR-Cas systems.
Lots of CRISPyness
Several themes emerged from the work. One is that some of the newly identified CRISPR systems appear to use Cas enzymes with never-before-seen domains or are fusions with known genes. Scientists have further characterized some of these, and have identified an enzyme that is more specific than currently used CRISPR enzymes, and an enzyme that cuts RNA that they propose could lead to an entirely new seventh type of CRISPR-Cas. discovered that they are structurally different enough to form a system.
A corollary of this theme is the collaboration of CRISPR arrays with enzymes with a variety of functions, not just nucleases (enzymes that cut DNA and RNA). Scientists have taken advantage of CRISPR’s remarkable gene targeting abilities by linking it to molecules such as other types of enzymes and fluorescent dyes. But it’s clear that evolution got there first.
As an example, FLSHclust has identified something called a transposase that is associated with two different types of CRISPR systems. Transposases are enzymes that help certain parts of DNA jump to other parts of the genome. CRISPR RNA-induced transfer has been seen before, but this is another example. A large number of proteins with different functions, including those with transmembrane domains and signaling molecules, were found to bind to CRISPR arrays, highlighting the mix-and-match nature of the evolution of these systems. Researchers also discovered a protein expressed by the virus that binds to CRISPR arrays and inactivates them. Essentially, the virus deactivates her CRISPR system, which evolved to protect against viruses.
In addition to discovering a novel protein associated with CRISPR arrays, the researchers also discovered other regularly spaced repeat arrays that are similar to but not CRISPR and are not associated with cas enzymes. They do not know what the function of these RNA-guided systems is, but they speculate that they may be involved in defense, similar to CRISPR.
The authors aimed to find “a catalog of RNA-guided proteins that will expand our understanding of the biology and evolution of these systems and provide a starting point for the development of new biotechnologies.” It looks like they achieved their goal. “This study reveals the unprecedented organizational and functional flexibility and modularity of the CRISPR system,” the researchers wrote, going on to conclude: The remaining candidates serve as resources for future exploration. ”
Article DOI: 10.1126/science.adi1910
