To find out what the gene does, researchers turn it off and watch for consequences. Genes often have multiple functions that vary with tissue and age. Some genes are essential for growth and stopping them early can have dire consequences that can make monitoring other functions impossible. To avoid this, researchers have been using a conditional gene inactivation that allows the gene to be turned off only in a specific tissue or at a later time in development, for example, in adulthood.
One of the systems used for conditional gene inactivation is Cre / lox. “It’s the gold standard for conditional gene inactivation in mice, but over time it has also become very important in other model organisms like zebrafish,” says Dr. Stefan Hans, CRTD researcher and author of the study. The Cre / lox system relies on an enzyme known as Cre recombinase, and special sequences known as lox. Lox sequences are inserted into the genome around the respective gene.
Cre recognizes the lox sequence and removes a gene trapped between them. In this way, the gene is turned off and will not be expressed in this cell. Over the years, the research community has developed many animal strains in which recombinase is only present in a specific tissue. With these Cre lines, it is possible to turn off the gene in only one tissue but not in the other tissues.
Although it is commonly used, surrounding a gene with a lox sequence has its drawbacks. “Inactivating a specific gene takes a lot of time and effort. It requires complex modifications of the genome and can take multiple generations of animals for a preparation to be ready for an experiment,” Dr. Hans explains. “Compared to the method we created, it’s very slow and arduous,” he adds.
Cre-Controlled CRISPR, a new method developed by a team of Dresden researchers led by Professor Michael Brand, combines the advantages of the Cre / lox system with the CRISPR / Cas9 gene clipper CRISPR / Cas9 is a relatively new method that has revolutionized the life sciences Quickly and lead to the 2020 Nobel Prize in Chemistry. Although generally easy to use, the CRISPR / Cas9 technology is not easily restricted to just one tissue, meaning that adapting it to inactivating the conditional gene takes time and effort.
“In a Cre-controlled CRISPR technology, we take advantage of the tissue-specific expression of Cre and the ease of gene editing for CRISPR / Cas9,” says Dr. Hans. “By combining the two methods, we created a version of CRISPR / Cas9 that is triggered by recombinase Cre. Using our method, researchers can still take advantage of the vast libraries of already established animal strains that express Cre in different tissues. But the CRISPR technology is controlled. In it with CRISPR, it removes the laborious genome editing process because it removes the necessity to surround the gene with lox sequences. In fact, it only takes one sequence to be added to the genome, regardless of the gene in question, ”Dr. Hans explains. Without the need for gene binding between lox sequences, Cre-Controlled CRISPR is faster and easier.
The Cre-controlled CRISPR technology is not only easier to establish, but, just like CRISPR / Cas9, it also provides the ability to turn off multiple genes simultaneously. Moreover, the CRTD researchers designed their method to facilitate subsequent analyzes of cells whose controlled CRISPR was used. Once CRISPR / Cas9 is on, cells are labeled with green fluorescent protein (GFP). Fluorescent proteins such as GFP are commonly used in the life sciences and provide countless methods for separating labeled cells from other cells for use in other experiments, for example, next-generation sequencing or others.
“Although we developed the CRISPR technology controlled by Cre as a proof of concept in zebrafish, the method is versatile and should be applicable to other model organisms as well,” Dr. Hans adds.
Stefan Hans, Daniela Zoller, Julian Hammer, Joanna Stock, Sandra Speys, Jocole Kesavan, Volker Crohne, Juan Sebastian Eguren, Diana Iscova, Andreas Petzold, Andreas Dahl and Michael Brand: CRISPR-controlled mutations provide quick and easy CRISPR. Zebrafish. Nature Communications (February 2021)
About the Center for Regenerative Therapy Dresden (CRTD)
The Center for Regenerative Therapy Dresden (CRTD) at TU Dresden is the academic home of scientists from more than 30 countries. Their mission is to discover the principles of cell and tissue regeneration and to utilize this to recognize, treat and reverse disease. CRTD connects the chair to the clinic, scientists and clinicians to pool expertise in stem cells, evolutionary biology, gene editing and regeneration toward innovative treatments for neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease, blood diseases such as leukemia, and metabolic diseases such as diabetes and retinal and bone diseases.
Since 2016, CRTD has been part of the central scientific unit “Center for Molecular and Cellular Bioengineering” (CMCB) at TU Dresden and plays a central role in the research priority area of health sciences, biomedicine and bioengineering at TU Dresden.
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About the DRESDEN-Concept Genome Center (DcGC)
DcGC is a joint sequencing facility for the core facility for deep sequencing at the Center for Molecular and Cellular Bioengineering (CMCB) at TU Dresden and the sequencing facility of the Max Planck Institute for Molecular Cell Biology and Genetics (MPI-CBG). It is one of four DFG-funded German competency centers for next-generation sequencing. DcGC is an amalgamation of the staff working for CMCB, MPI-CBG, as well as Center for Systems Biology Dresden (CSBD). DcGC consists of three platforms focusing on the techniques of long read sequencing, single cell sequencing and short read sequencing, covering experimental workflow and bioinformatics analysis.