CRISPR-Cas9 makes it easy to knock out or tweak a single gene to determine its effect on an organism or cell, or even another gene. But what if you could perform several thousand experiments at once, using CRISPR to tweak every gene in the genome individually and quickly see the impact of each?
A team of scientists has developed an easy way to do just that, allowing anyone to profile a cell, including human cells, and rapidly determine all the DNA sequences in the genome that regulate the expression of a specific gene.
While the technique will mostly benefit basic researchers who are interested in tracking the cascade of genetic activity -- the genetic network -- that impacts a gene they're interested in, it will also help researchers quickly find the regulatory sequences that control disease genes and possibly find new targets for drugs.
"A disease where you might want to use this approach is cancer, where we know certain genes that those cancer cells express, and need to express, in order to survive and grow," said the senior author. "What this tool would let you do is ask the question: What are the upstream genes, what are the regulators that are controlling those genes that we know about?"
Those controllers may be easier to target therapeutically in order to shut down the cancer cells.
The new technique simplifies something that has been difficult to do until now: backtrack along genetic pathways in a cell to find these ultimate controllers. The authors published the details of their technique in the journal Science.
Since the advent of CRISPR-Cas9 gene-editing eight years ago, researchers who want to determine the function of a specific gene have been able to precisely target it with the Cas9 protein and knock it out. Guided by a piece of guide RNA complementary to the DNA in the gene, the Cas9 protein binds to the gene and cuts or, as with CRISPR interference (CRISPRi), inhibits it.
In the crudest type of assay, the cell or organism either lives or dies. However, it's possible to look for more subtle effects of the knockout, such as whether a specific gene is turned on or off, or how much it's turned up or down.
Today, that requires adding a reporter gene -- often one that codes for a green fluorescent protein -- attached to an identical copy of the promoter that initiates expression of the gene you're interested in. Since each gene's unique promoter determines when that gene is expressed, if the Cas9 knockout affects expression of your gene of interest, it will also affect expression of the reporter, making the culture glow green under fluorescent light.
Nevertheless, with 6,000 total genes in yeast -- and 20,000 total genes in humans -- it's a big undertaking to tweak each gene and discover the effect on a fluorescent reporter.
"CRISPR makes it easy to comprehensively survey all the genes in the genome and perturb them, but then the big question is, How do you read out the effects of each of those perturbations?" the author said.
This new technique, which the authors call CRISPR interference with barcoded expression reporter sequencing, or CiBER-seq, solves that problem, allowing these experiments to be done simultaneously by pooling tens of thousands of CRISPR experiments. The technique does away with the fluorescence and employs deep sequencing to directly measure the increased or decreased activity of genes in the pool. Deep sequencing uses high-throughput, long-read next generation sequencing technology to sequence and essentially count all the genes expressed in the pooled samples.
"In one pooled CiBER-seq experiment, in one day, we can find all the upstream regulators for several different target genes, whereas, if you were to use a fluorescence-based technique, each of those targets would take you multiple days of measurement time," the author said.
CRISPRing each gene in an organism in parallel is straightforward, thanks to companies that sell ready-made, single guide RNAs to use with the Cas9 protein. Researchers can order sgRNAs for every gene in the genome, and for each gene, a dozen different sgRNAs -- most genes are strings of thousands of nucleotides, while sgRNAs are about 20 nucleotides long.
The team's key innovation was to link each sgRNA with a unique, random nucleotide sequence -- essentially, a barcode -- connected to a promoter that will only transcribe the barcode if the gene of interest is also switched on. Each barcode reports on the effect of one sgRNA, individually targeting one gene out of a complex pool of thousands of sgRNAs. Deep sequencing tells you the relative abundances of every barcode in the sample -- for yeast, some 60,000 -- allowing you to quickly assess which of the 6,000 genes in yeast has an effect on the promoter and, thus, expression of the gene of interest. For human cells, a researcher might insert more than 200,000 different guide RNAs, targeting each gene multiple times.
"This is really the heart of what we were able to do differently: the idea that you have a big library of different guide RNAs, each of which is going to perturb a different gene, but it has the same query promoter on it -- the response you are studying. That query promoter transcribes the random barcode that we link to each guide," the author said. "If there is a response you care about, you poke each different gene in the genome and see how the response changes."
If you get one barcode that is 10 times more abundant than any of the others, for example, that tells you that that query promoter is switched on 10 times more strongly in that cell. In practice, the authors attached about four different barcodes to each guide RNA, as a quadruple check on the results.
"By looking more directly at a gene expression response, we can pick up on a lot of subtlety to the physiology itself, what is going on inside the cell," the author said.
In the newly reported experiments, the team queried five separate genes in yeast, including genes involved in metabolism, cell division and the cell's response to stress. While it may be possible to study up to 100 genes simultaneously when CRISPRing the entire genome, for convenience, researchers would limit themselves to four or five at once.
https://news.berkeley.edu/2020/12/10/using-crispr-new-technique-makes-it-easy-to-map-genetic-networks/
https://science.sciencemag.org/content/370/6522/eabb9662
Mapping genetic networks using CRISPR
- 1,606 views
- Added
Edited
Latest News
A new tool for neurological…
By newseditor
Posted 26 Jun
Psychosocial experiences ar…
By newseditor
Posted 25 Jun
How a microbe and a prebiot…
By newseditor
Posted 24 Jun
Slowing inflammation may bo…
By newseditor
Posted 24 Jun
Cellular senescence in norm…
By newseditor
Posted 24 Jun
Other Top Stories
Vascular endothelial growth factor receptor gets help in the angiog…
Read more
A new mode of drug resistance to emerging therapies in prostate cancer
Read more
New mechanism for aspirin's role in cancer prevention
Read more
Liver cancer metabolic profile
Read more
Mutations in key cancer protein suggest new route to treatments
Read more
Protocols
BicemuS: A new tool for neu…
By newseditor
Posted 26 Jun
Deciphering spatial domains…
By newseditor
Posted 23 Jun
High-throughput volumetric…
By newseditor
Posted 21 Jun
Bioengineered human colon o…
By newseditor
Posted 14 Jun
Development of an efficient…
By newseditor
Posted 12 Jun
Publications
Nicotinamide metabolism fac…
By newseditor
Posted 26 Jun
Tonic type 2 immunity is a…
By newseditor
Posted 26 Jun
An unexpected role for the…
By newseditor
Posted 25 Jun
Coordinated action of a gut…
By newseditor
Posted 25 Jun
Safety of non-replicative a…
By newseditor
Posted 25 Jun
Presentations
Arabidopsis CaLB1 undergoes…
By newseditor
Posted 26 Jun
Myelin plasticity in the ve…
By newseditor
Posted 10 Jun
Hydrogels in Drug Delivery
By newseditor
Posted 12 Apr
Lipids
By newseditor
Posted 31 Dec
Cell biology of carbohydrat…
By newseditor
Posted 29 Nov
Posters
A chemical biology/modular…
By newseditor
Posted 22 Aug
Single-molecule covalent ma…
By newseditor
Posted 04 Jul
ASCO-2020-HEALTH SERVICES R…
By newseditor
Posted 23 Mar
ASCO-2020-HEAD AND NECK CANCER
By newseditor
Posted 23 Mar
ASCO-2020-GENITOURINARY CAN…
By newseditor
Posted 23 Mar