Micro-recording devices

Bacteria were forced to write work reports using CRISPR

Anna Kaznadzei, Naked Science

Scientists have figured out how to use the CRISPR-Cas system in order to force cells to sequentially record the "history" of biological signals into their own genome. This will allow tracking, for example, changes in metabolism and the level of gene expression, as well as distinguishing cell lines by the nature of the biochemical processes taking place in them. The study is published in the journal Science (Sheth et al., Multiplex recording of cellular events over time on CRISPR biological tape).

The CRISPR-Cas system is now widely used for genomic editing, but in nature it is needed by bacteria, in particular, in order to fight foreign (for example, viral) genetic material. The system has the ability to "write" pieces of foreign DNA into its own genome, so that then, using these records as templates, it can find such DNA in the cytoplasm and cut it, thus fighting pathogens. The template sites are called "spacers", and they are arranged in a strictly regular order – new spacers are always added to the CRISPR cassette in the genome from the same end.

Scientists took advantage of this feature of the CRISPR system and developed a technology called TRACE (from the English word "track"; the abbreviation stands for "Temporary Recording in Arrays by CRISPR Expansion"). The technology is based on the following principle: small sections of DNA, which are called trigger spacers, are "recorded" in a CRISPR cassette under the influence of certain signals, and when there are no signals, instead, ordinary, "reference" spacers are recorded there at a pace familiar to the cell. After that, after reading the tape, you can detect when and which spacer was embedded in it, and thus understand what changes occurred in the cell over time.

Drawings from the press release of Columbia University Medical Center
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Trigger spacers appear in the cell due to special artificial plasmids (circular DNA molecules carrying several genes that can replicate themselves). They were designed in such a way that, with an increase in the level of a given signaling molecule (initially it was IPTG, isopropyl-β-D-1-thiogalactopyranoside), the expression of the protein responsible for plasmid replication increased in their environment, which, in turn, significantly increased the number of plasmids of this type in the cell. After making sure that this system works, the scientists also made other plasmids, with genes encoding the Cas1 and Cas2 proteins. These proteins are responsible for embedding new spacers into CRISPR. The expression of these genes was also controlled by certain signaling molecules (anhydrotetracycline). The inclusion of the expression of genes of the second type of plasmids (i.e., the synthesis of Cas1 and Cas2) caused the successful embedding of a significant number of spacers from plasmids of the first type into CRISPR cassettes, which became very numerous in the cell when activated by IPTG.

Spacers were embedded in cassettes at different speeds, and to account for these differences, scientists created an analytical model that allows calculating the probability of embedding certain spacers in certain positions when a signal occurs or is absent. Having tested the technology on Escherichia coli, they were convinced that the TRACE system allows you to successfully track the addition and exclusion of IPTG in time. After that, they expanded the experiment by forcing the system in three different types of cells to respond to three different types of biological signals for three days. Copper, trehalose and fucose were used as signals, which appeared in the medium at different times in different order. The system literally recorded the corresponding sequence in CRISPR, and then this record could be read using sequencing and decoded using an analytical model.

Scientists believe that a new system that sequentially records what is happening in a cell can be used to temporarily assess changes in gene expression and metabolic processes, including in environments where it is inconvenient to track these processes in another way – for example, in animal gut microbiomes or in marine bacterial communities. The recording speed can also be edited by changing the operation of Cas proteins – scientists are going to further improve the system, making it more accurate and sensitive. Having achieved a certain degree of sensitivity, it will be possible to record changes even inside individual cells, they believe.

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