BD ACCURI NEWS
A Cas9 toolkit for yeast
Maren Wehrs is a graduate student at the Technical University of Braunschweig, Germany, pursuing her research in a collaborative arrangement with Dr. Aindrila Mukhopadhyay’s lab at the Lawrence Berkeley National Laboratory in Berkeley, CA. Her coauthored paper describing a Cas9-based toolkit for a yeast strain was a recent BD Accuri News Publication Pick. Ms. Wehrs told us why genetic manipulation in yeast is important and how their BD Accuri™ flow cytometer helped them develop the toolkit.
Read the interview »
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A closer look at endogenous CRISPR-Cas systems
CRISPR-Cas systems like those explored in this month’s interview have achieved such notoriety as a genome editing tool that we sometimes forget that they evolved as endogenous prokaryotic immune systems, and their potential usefulness extends in many directions. In a BD Biosciences webinar on molecular biology applications of personal flow cytometry, Dr. Chase Beisel of North Carolina State University described his laboratory’s research program to understand CRISPR-Cas biology and exploit CRISPR-Cas systems. He also explained why he and his colleagues are “heavy users” of their BD Accuri flow cytometer.
As Dr. Beisel explained, CRISPR-Cas systems are naturally adaptive immune systems found in bacteria and archaea. Their role is to fend off foreign invaders like plasmids and bacteriophages. Unlike our own adaptive immune systems, they use RNA as the recognition element, looking for complementary nucleic acids—primarily DNA. When it finds them, it instructs the system to cleave and degrade the DNA. It's a great way to get rid of these invaders.
In the webinar, Dr. Beisel explored Type I CRISPR-Cas systems, which rely on multiple proteins and have a unique ability to cleave and degrade DNA (and are far more common in nature than the Type II CRISPR-Cas system and its Cas9 protein used in genome editing). For example, Type I-E CRISPR-Cas systems found in E. coli use a Cas protein complex called Cascade to process the CRISPR array, use the RNA to bind to target sequences, and then recruit Cas3 to nick and degrade the displaced DNA strand. The Beisel lab showed that eliminating Cas3 still allowed Cascade to bind its DNA target, resulting in blocked transcription at that site.
Figure 1 shows the workflow for an experiment that attempted to co-opt this system as a gene-silencing method. A BD Accuri flow cytometer was used to measure green fluorescent protein (GFP) as a reporter of gene expression.
Figure 1. Workflow for transcriptional repression studies
Figure courtesy of Dr. Chase Beisel, North Carolina State University.
The results are shown in Figure 2. Tn and NTn represent the different locations at which transcription was targeted by blocking RNA polymerase. Flow cytometry analysis of GFP expression showed up to 100-fold (or more) repression of gene expression compared to a non-targeting control, primarily when targeting the promoter (as opposed to coding regions). The histograms at bottom right, output from BD Accuri™ software, show differential repression for three representative targeting locations. Gene silencing was uniform (rather than bimodal) across the population—a flow cytometry finding that would not have been possible using bulk characterization techniques.
Figure 3 shows how Dr. Beisel’s group then exploited this gene-silencing method to turn off genes responsible for the catabolism of one of four different sugars that E. coli can grow on, targeting four different gene promoters. Each modified strain was then grown in media containing that sugar. The knockdown resulted in roughly 100- to 1,000-fold repression in growth, reflected in the top bar graph and the left column of histograms. They also created an array of all four targeted spacers, which effectively repressed growth in all four media (bottom bar graph, right histograms), even though the bacteria grew normally in two control sugars whose promoters were not targeted. Dr. Beisel’s group has created a powerful technique for turning off microbial genes either individually or combinatorially, which can be useful in understanding their basic biology, in high-throughput screens and to engineer their transcriptomic landscape.
Also in the webinar, BD scientist Dr. Mirko Corselli presented data on the use of flow cytometry to optimize transfection efficiency in mammalian cells and to detect fluorescent proteins in bacteria. Both presentations demonstrated that personal flow cytometry can be a powerful and invaluable tool in molecular and microbiology research and discovery.
Tips & Tricks
How can you prevent contamination in the sample injection probe (SIP), flow cell and sheath fluid bottle of your BD Accuri flow cytometer?
With the BD Accuri C6 Plus, there is no separate decontamination procedure. Just follow the steps in the BD Accuri™ C6 Plus System User’s Guide to perform a SIP clean, clean the fluidics and clean the outside of the instrument.
With the BD Accuri™ C6 flow cytometer, after each experiment, prepare a tube of decontamination solution, diluted to working concentration according to the package instructions. Run for 2 minutes on Fast speed. BD Accuri™ Decontamination Concentrate Solution (Cat. No. 653154 or 653155) is designed for this purpose. Follow by running filtered, deionized water for 2 minutes on Fast speed to flush out cellular debris and prevent clogging. If sheath fluid remains in the fluidics bottle for more than two days, add BD Accuri™ Bacteriostatic Concentrate Solution (Cat. No. 653156) to prevent bacterial contamination.
This section highlights interesting recent articles that describe research using BD Accuri flow cytometers.
Skin cancer therapeutics
Pal HC, Katiyar SK. Cryptolepine, a plant alkaloid, inhibits the growth of non-melanoma skin cancer cells through inhibition of topoisomerase and induction of DNA damage. Molecules. 2016;21:1758. PubMed
Off-target transient transfection assays
Masuda T, Wan J, Yerrabelli A, et al. Off target, but sequence-specific, shRNA-associated trans-activation of promoter reporters in transient transfection assays. PloS One. 2016;11:e0167867. PubMed
Lim SI, Lukianov CI, Champion JA. Self-assembled protein nanocarrier for intracellular delivery of antibody. J Control Release. 2017;249:1-10. PubMed
LED bacterial inactivation
Kim MJ, Yuk HG. Antibacterial mechanism of 405-nanometer light-emitting diode against salmonella at refrigeration temperature. Appl Environ Microbiol. 2017;83:e02582-16. PubMed
Meeting – December 2–6, 2017 – Philadelphia, PA
ASCB/EMBO 2017 (American Society for Cell Biology/European Molecular Biology Organization) »
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