June 11, 2016

100 bits recorded in e coli bacteria using CRISPR editing

Utilizing the bacterial CRISPR/Cas adaptive immune system, researchers at Harvard have developed a method for permanently recording molecular events in living cells

Before this experiment, the most information any scientist had ever permanently uploaded into a living cell was 11 bits of information. That's a mere 11 zeros and ones of binary data, and less information than your computer requires to code for two alphabetic letters. This new technique expanded this record to roughly 100 bytes of data. Shipman says the 100 bytes his team demonstrated is nothing near the limit. Certain cells, like the microorganism Sulfolobus tokodaii would have room for more than 3,000 bytes of data. And with synthetic engineering, it's not hard to imagine certain specially designed hard-drive bacteria with vastly expanded regions of their genetic code, able to rapidly upload vast amounts of data.

The CRISPR/Cas system works by snipping short DNA elements from the genomes of infecting viruses, integrating those elements into the bacterium’s genome (at the CRISPR locus), and using the RNAs produced from the integrated elements to direct destruction of the corresponding virus. In essence, the bacterium keeps a DNA account of its viral foes, and uses it against them.

Integration of these viral DNA elements—or oligomers—into the CRISPR locus is nonrandom: the most recent viral elements are consistently integrated ahead of older viral elements in the array. Harvard’s George Church and colleagues considered that this temporal ordering of integration could form the basis of a molecular recording device. If defined synthetic DNA oligomers could be integrated into CRISPR loci just as viral elements are, then sequencing the cells’ CRISPR loci would provide a log of which oligomers the cells had been exposed to and when, the researchers reasoned.

The team used an E. coli strain that contained a CRISPR DNA locus and a stripped-down version of the Cas protein machinery. The minimal machinery consisted of inducible versions of Cas1 and Cas2—enzymes required for integrating the DNA oligomers—but lacked all the Cas machinery required for virus destruction. The researchers found that, by introducing specific synthetic DNA sequences into these cells in a timed manner (different oligomers on different days, for example), the resulting sequences of the CRISPR loci did indeed accurately reflect the order in which the oligomers had been introduced.

Using directed evolution, the team went on to create new versions of Cas1 and Cas2 that could integrate oligomers in a subtly different and discernable way (though still temporally ordered) to that of wildtype Cas1 and 2. Putting these modified Cas enzymes under the control of a different inducer allowed the team to record DNA events in two different modes—depending on which versions of Cas1 and 2 were operational.

“Essentially, we’re measuring concentrations of nucleic acids,” said Church. “Ideally it would be messenger RNAs but in this case it is DNA. . . . This is a proof of concept on the way to other things,” he added.

Church suggested, for example, that if a CRISPR/Cas system were to be combined with a reverse transcriptase—an enzyme that converts RNA to DNA—in cells or animals, it could be used to provide a record of which messenger RNAs are expressed, when.

Another possibility, suggested Arkin, is to use CRISPR/Cas-engineered bacteria to provide information about the other microorganisms present in an environment—be that the soil, the human gut, or wherever.

“[The bacteria] could kill a few neighboring [bugs], secrete an enzyme that cleaved their DNA, and express a competence system to take that DNA in,” Arkin said. “That sounds insane, but there are bacteria who do that naturally,” he added. The foreign microbial DNA could then be incorporated and logged at the bacteria’s CRISPR locus, he explained.

Science - Molecular recordings by directed CRISPR spacer acquisition


The ability to write a stable record of identified molecular events into a specific genomic locus would enable the examination of long cellular histories and have many applications, ranging from developmental biology to synthetic devices. We show that the type I-E CRISPR-Cas system of E. coli can mediate acquisition of defined pieces of synthetic DNA. We harnessed this feature to generate records of specific DNA sequences into a population of bacterial genomes. We then applied directed evolution to alter the recognition of a protospacer adjacent motif by the Cas1-Cas2 complex, which enabled recording in two modes simultaneously. We used this system to reveal aspects of spacer acquisition, fundamental to the CRISPR-Cas adaptation process. These results lay the foundations of a multimodal intracellular recording device.

SOURCE -The Scientist, Popular Mechnanics, Science

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