NEW YORK (GenomeWeb) – Building on a method that uses toothless Cas9 proteins tagged with a fluorescent protein to track chromosomes, scientists have created a multicolor CRISPR/Cas9-based system to label inter-and intrachromosomal loci in the cell nucleus.
The method provides high-resolution estimates of spatial and temporal relationships between loci in live cells and could reveal the contributions of chromosomal mechanics to diseases like cancer and aneuploidy.
Led by Hanhui Ma and Thoru Pederson of the University of Massachusetts Medical School, the scientists reported their method earlier this month in the Proceedings of the National Academy of Sciences. The tool promises to be useful "for probing dynamic interactions of intrachromosomal and interchromosomal domains during cell cycle progression, during epigenetic regulation, or in response to cellular stimuli," the authors wrote.
The method features green, red, and blue fluorescent proteins fused to three different Cas9 variants from three separate bacterial species: Streptococcus pyogenes, Neisseria meningitidis, and Streptococcus thermophilus. "Different Cas9 enzymes can recognize different guide RNAs and tag to different regions of the genome," Ma explained, allowing the system to be multicolored. All three enzymes have been used for editing and gene regulation in human cells.
"This is a kind of dead Cas9," Ma told GenomeWeb."It's not active, but the advantage is this Cas9 is still able to bind the genome as long as it has guides specific to the loci."
The fluorescent tags, put in place with the use of guide RNAs, allowed the scientists to track the movement of chromosomal loci in time and space. Using a standard fluorescence microscopy system, they were able to detect two pairs of chromosomal loci that lie 1.9 or 2.0 Mbp apart in the DNA physical map.
Ma sees chromosome tracking as yet another research application of CRISPR/Cas9, in addition to direct genome editing and fusing it to transcription factors to ramp levels of gene expression up or down. "Many scientists want to use this to track what's happening in disease," Ma said, but added that scientists could use it to study a variety of events in the cell.
One specific application is to view translocations in live cells, which can play a fundamental role in the development of cancer. "When the translocation steps happen, what happens to the cells? Is there trouble in cell division? They want to see what really happens in the cells," he said, and multicolor CRISPR/Cas9 tagging can help them do just that.
Though CRISPR/Cas9-based tagging of chromosomes with fluorescent proteins was first established by scientists at the University of California, San Francisco, led by Baohui Chen and Bo Huang, the UMass scientists improved it by adding multiple colors, allowing greater resolution of loci close together on a chromosome.
Ma and his colleagues had previously worked with transcription activator-like effectors to track chromosomal loci, but have focused on using CRISPR/Cas9 because it can be programmed to target a broad range of genomic targets.
The authors noted that their method "may provide a new tool in the study of interphase DNA compaction in live cells, particularly for examining genomic regions that may have unusual chromatin structure."
The method may also help elucidate the inner lives of cells, including gene expression, which can depend on the unfurling and compaction of DNA. "If you have a method to look where a gene is, sometimes you can predict whether the gene is on or off, based on the location," Ma said. "If a gene is at the nuclear periphery, much of the time it will be silenced." The method's ability to resolve is especially necessary for DNA compaction associated with lower levels of gene expression.
Ma said he and his colleagues have fielded several collaboration requests from labs who want to use the tracking method to study chromosomal errors during cell division related to cancer, such as missing chromosomes or missegregations. "If they can track the errors, they'll know what's happening in the disease," Ma said.
Ma was reluctant to specify other applications for the technique because he said he is in the process of filing a patent, but said there is a potential clinical application in determining chromosome number.
Though Ma said the chromosomal tagging might not necessarily be faster than current methods of determining aneuploidy, it's a potential clinical use.