April 02, 2015
Editing is an essential part of producing good writing. You might cringe when your masterpiece comes back covered in red ink, but in the end your paper is better for it.
These days, computers have made editing much, much faster and easier than using ink on paper. Some of us remember when cutting and pasting literally mean cutting the piece of paper on which your manuscript was typed and pasting the sections in a different order!
The same kind of revolution has happened when it comes to editing genomes. A seemingly obscure system used by bacteria to defend against invading phages, discovered in the 1950s, led to the development of restriction enzymes as the reagents that have enabled virtually all modern molecular biology. And now, an equally “obscure” system of bacterial immunity has opened the door to genome editing that is as precise and nearly as fast as your thesis advisor using Microsoft Word to edit your dissertation (maybe faster!).
This bacterial system is called CRISPR/Cas. Bacteria use it to defend against the foreign DNA—from infecting phages, bacteria that transfer plasmids via conjugation, or other sources—that constantly assaults them. And now scientists are using it to edit the genomes of most any beast they want, including our favorite yeast.
What makes this technique so powerful is that it is easily programmable. You can make virtually any sequence change that you wish, and even more impressively, make lots of changes all at once. CRISPR/Cas may be one of those technological leaps that changes everything.
The incredible potential of this approach, and the ethics of its use in humans, are hot topics right now. But while researchers and philosophers are hammering out guidelines for using the CRISPR/Cas system in larger organisms, yeast researchers are free to forge ahead and edit the S. cerevisiae genome to their hearts’ content. This has now been made much easier with the availability of a whole toolkit, created at the Delft University of Technology and described in a new paper by Mans et al.
The yeast system has three essential components. The first is a plasmid that will express a specific guide RNA (gRNA). The gRNA leads a nuclease to the right place in the genome. Mans and colleagues made a whole set of plasmids, with different nutritional and antibiotic resistance markers, that can express one or more gRNAs.
The second component of the system is the nuclease that binds to the gRNA and makes a double-stranded cut in the target DNA. The researchers used the Cas9 nuclease from Streptococcus pyogenes, and engineered a set of yeast strains that had the cas9 gene stably integrated into a chromosome and expressed from a strong yeast promoter.
The third component is one or more repair fragments: pieces of DNA that specify the modified sequence that the researcher wants to engineer into the genome.
So, for example, if a scientist wants to delete a gene precisely, she can create a gRNA that targets the gene, and then co-transform a Cas9-expressing yeast strain with both the plasmid expressing the gRNA and a repair fragment that corresponds to the gene’s upstream and downstream flanking sequences, fused together. When the gRNA is expressed in the transformant, it leads Cas9 to the gene, where it makes a double-stranded cut at a precise position in the DNA.
Now the researcher can let yeast do the rest of the work. S. cerevisiae has a powerful homologous recombination system, and it’s greatly stimulated by double-stranded breaks in DNA.
After Cas9 cuts the gene, yeast will repair the break, using as a template the repair fragment that the researcher designed. In this case, the upstream and downstream sequences will recombine with the homologous sequences in the chromosome, but since the coding sequence is missing from the repair fragment, the resulting strain will have a precise deletion of the gene of interest.
This example illustrates one of the simplest uses of the technique. Mans and colleagues tried successively more complicated tasks and were able to accomplish some amazing feats.
They were able to precisely delete six genes in one step by transforming with repair fragments for all six, along with three plasmids that each expressed two gRNAs. Also in one step, they replaced one yeast gene with six Enterococcus faecalis genes encoding subunits of the pyruvate dehydrogenase complex and other enzymes in the pathway. The E. faecalis genes were specified on six overlapping repair fragments that were cotransformed into the strain.
The CRISPR/Cas9 system designed by Mans and colleagues can do much more than gene deletions and replacements. By designing repair fragments specifying particular mutations, the method can also be used to create point mutations or other modifications.
For this technique to work, it’s important that there are no mismatches between the gRNA sequence and the chromosomal target sequence, and other sequence characteristics can influence the efficiency of the method. So the authors created an online tool that helps researchers select optimal Cas9 targets in regions of interest and design gRNA sequences. Since they incorporated the sequences of 33 different S. cerevisiae strains into the tool, researchers can specify a strain and retrieve information on the best sequences for targets and gRNAs for their gene(s) of interest based on sequences found in that particular strain.
Importantly, the CRISPR/Cas9 technique allows researchers to make multiple changes in a single step. This is a big advantage, since transformation itself can be mutagenic. For example, a strain that has been commonly used to investigate the function of hexose transporters was engineered to carry multiple deletions in the conventional manner, using successive rounds of transformation and selection. Its genomic sequence, which was recently determined, reveals that its genome is a complete mess, with many rearrangements and deletions.
With the development of this toolkit, editing the S. cerevisiae genome is beginning to be almost as easy as editing a text document. And since Mans and colleagues have made all of the strains, plasmids, and online tool freely available to the world, everyone will be able to take advantage of them. Just think of the stories that yeast researchers will be able to write!
Way before this became a powerful tool for researchers, it was a very cool immune system for bacteria, allowing them to defend against assaults from foreign invaders.
Bacteria collect foreign DNA sequences from invaders that threaten them, just like collecting mug shots of notorious criminals. They store these mug shots in a special place in their genome, integrated between blocks of a repeated sequence. (The CRISPR acronym refers to these repeats.) This region is transcribed, and the RNA is chopped into pieces containing individual mug shots. Because these mug shots, called crRNAs, are complementary to the DNA sequences of invaders, they can recognize and hybridize with those invading sequences.
Bacteria have an additional small RNA, the tacrRNA, that binds to both the crRNA and to a CRISPR-associated (Cas) nuclease. This forms a RNA-DNA-protein complex on the foreign DNA and allows the nuclease to do its work, cutting both strands of the DNA and neutralizing the invader.
To use this system for genome engineering, scientists have fused the two RNAs of the bacterial system into a single RNA, the guide RNA (gRNA). It contains both the mug shot (with sequences complementary to the target) and the RNA sequence that recruits the nuclease. The gRNA, a Cas nuclease, and a repair fragment are the essential components of the system.
by Maria Costanzo, Ph.D., Senior Biocurator, SGD
Categories: Research Spotlight