The physicist’s guide to one of biotechnology’s hottest new topics: CRISPR-Cas

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) constitute a multi-functional, constantly evolving immune system in bacteria and archaea cells. A heritable, molecular memory is generated of phage, plasmids, or other mobile genetic elements that attempt to attack the cell. This memory is used to recognize and interfere with subsequent invasions from the same genetic elements. This versatile prokaryotic tool has also been used to advance applications in biotechnology. Here we review a large body of CRISPR-Cas research to explore themes of evolution and selection, population dynamics, horizontal gene transfer, specific and cross-reactive interactions, cost and regulation, non-immunological CRISPR functions that boost host cell robustness, as well as applicable mechanisms for efficient and specific genetic engineering. We offer future directions that can be addressed by the physics community. Physical understanding of the CRISPR-Cas system will advance uses in biotechnology, such as developing cell lines and animal models, cell labeling and information storage, combatting antibiotic resistance, and human therapeutics.

The size of the immune repertoire of bacteria

“…The CRISPR-Cas mechanism for immunity has three stages [2,3]. Following a first encounter with a virus, after a successful defense through another mechanism or if the virus is ineffective for some reason [4], some of the Cas proteins recruit pieces of viral DNA and integrate these "spacers" into an array separated by palindromic repeated sequences in the CRIPSR locus of the bacte-rial genome.…”

The size of the immune repertoire of bacteria

“…The CRISPR-Cas mechanism for immunity has three stages (2,3). Following a first encounter with a virus, after a successful defense through another mechanism or if the virus is ineffective for some reason (4), some of the Cas proteins recruit pieces of viral DNA and integrate these spacers into an array separated by palindromic repeated sequences in one of several CRISPR loci (5) of the bacterial genome.…”

“…A clue may be offered in the fact that older spacers often correspond to evolutionarily conserved regions in the genomes of persistent viruses (3,11,(20)(21)(22). In fact, even if an older spacer is not a perfect match to a new invader, it may efficiently prime the acquisition of new immunity (23)(24)(25)(26)(27)(28)(29)(30).…”

CRISPR recognizes as many phage types as possible without overwhelming the Cas machinery

“…Reprinted from ref. 13, which is licensed under CC BY 3.0. spacers due to the selective pressure for targeting dominant phage genotypes (14), as had been observed in studies of environmental strains. A population dynamics model showed that a less diverse CRISPR spacer distribution that is focused on the most effective spacers evolved when phage had multiple protospacers that differ in their effectiveness, although a diverse spacer locus evolved when phage protospacers differed in their ease of acquisition (15).…”

“…Conversely, if the size is above the optimal value, there are so many distinct potential spacers that the requirement of d c Cas/crRNA complexes per phage type is not able to be met. This penalty addresses the biochemical requirement for efficient use and low energy consumption of Cas proteins (13).…”