Get­ting CRISPR

by Merry

When you're out­num­bered five or ten to one, strat­egy counts. Bac­te­ria and ar­chaea are mas­ter strate­gists, thriv­ing de­spite the om­nipresent hordes of phage. For some time, it has been known that prokary­otic cells have ploys for block­ing phage ad­sorp­tion, pre­vent­ing DNA in­jec­tion, rec­og­niz­ing and cleav­ing the in­com­ing DNA, and oth­er­wise abort­ing the in­fec­tion. Re­cently, an­other po­tent – and likely very an­cient – mech­a­nism of de­fense has been dis­cov­ered.

Phages on the sur­face of an Es­cherichia co­li cell de­liver ge­netic ma­te­r­ial into the bac­terium. © Eye of Sci­ence

In 1987, re­searchers re­ported find­ing that the E. coli genome con­tains some­thing unan­tic­i­pated, namely clus­ters of regu­larly inter­spaced short palin­dromic repeats, now known as CRISPRs. Since then, CRISPRs have been found all over – in 40% of all se­quenced bac­te­r­ial genomes, 90% of ar­chaeal genomes. A CRISPR is an ar­ray made up of a se­ries of di­rect re­peats al­ter­nat­ing with short in­ter­ven­ing re­gions ("spac­ers"). The num­ber of CRISPR ar­rays varies from or­gan­ism to or­gan­ism, as does the num­ber of re­peats. The re­peats within each ar­ray are al­most al­ways iden­ti­cal; the in­ter­ven­ing re­gions vary. Strong in­ter­est in CRISPRs was aroused when it was found that the se­quences of some spac­ers matched se­quences in known phages or plas­mids. (For an ex­cel­lent re­cent re­view, click here.)

Di­a­gram of a CRISPR ar­ray. CAS genes are CRISPR-as­so­ci­ated genes that en­code the CRISPR en­zy­matic ma­chin­ery. Source

Re­cently, Bar­ran­gou and col­leagues found that CRISPR sys­tems can pro­vide re­sis­tance to phage in­fec­tion in Strep­to­coc­cus ther­mophilus. They had good rea­sons for choos­ing S. ther­mophilus, as this is the work­horse of the dairy in­dus­try, used widely for the pro­duc­tion of cheese and yo­gurt. Since phage in­fec­tion has been an on­go­ing prob­lem for that in­dus­try, nu­mer­ous phage-re­sis­tant strains are avail­able, as well as genome se­quences for more than a dozen in­fect­ing phages. These re­searchers found that re­sis­tant strains have addi­tional in­ter­ven­ing re­gions (spac­ers) in one of their CRISPR ar­rays. Next step: they chal­lenged the phage-sen­si­tive strain with ei­ther of two phages, or with both si­mul­ta­ne­ously, and re­cov­ered nine new phage-re­sis­tant mu­tants. In every case the mu­tant had ac­quired be­tween one and four new in­ter­ven­ing re­gions. Star­tlingly, the se­quences of those re­gions were de­rived from the phage genomes.

When the se­quence of an in­ter­ven­ing re­gion was iden­ti­cal to a phage se­quence, the mu­tant was re­sis­tant to in­fec­tion by that phage, and re­sis­tance in­creased with the num­ber of such re­gions ac­quired. If even just one nu­cleotide did not match, re­sis­tance was lost. Thus it is the in­ter­ven­ing re­gions that pro­vide the speci­ficity – the busi­ness end of the ar­rays. One can think of this pheno­menon as a case of in­her­ited adap­tive im­mu­nity. CRISPRs add to the va­ri­ety of ways that we see genomes chang­ing in a reg­u­lated man­ner. Chro­mo­so­mal DNA, once the staid repos­i­tory of pro­tein-en­cod­ing in­for­ma­tion, has evolved into "the dy­namic genome."

Play­ground see­saw in Poland. Credit: Mo­hylek

This fast-paced arms race be­tween phage and prokar­yote host con­tin­ues. The ac­qui­si­tion of new in­ter­ven­ing re­gions by po­ten­tial host cells is rapid (with the old­est ones be­ing deleted to keep the size of CRISPR ar­rays in bounds). En­tire CRISPR sys­tems have been ac­quired by hor­i­zon­tal gene trans­fer. Phage evade the new de­fense by mu­ta­tions (ei­ther nu­cleotide sub­sti­tu­tions or dele­tions) in the cor­re­spond­ing se­quences, thus trump­ing that par­tic­u­lar CRISPR se­quence. The ad­van­tage see­saws back and forth. You might well be think­ing that phage, also be­ing ter­rific strate­gists, have evolved some ways to in­ac­ti­vate the host's CRISPR sys­tem. In­deed, the first sug­ges­tive ob­ser­va­tions have al­ready been re­ported.

The in­trigu­ing story, which is just start­ing to be de­ci­phered, is how CRISPRs work, i.e., how their in­for­ma­tion is trans­mit­ted. Al­though the ex­act mech­a­nism by which they con­fer im­mu­nity is not known, there is ev­i­dence sug­gest­ing a gene-si­lenc­ing mech­a­nism sim­i­lar to the RNAi path­way in eu­kary­otes. Since many of the in­ter­ven­ing re­gions are ho­mol­o­gous to host chro­mo­so­mal DNA, CRISPR might also play a role in reg­u­lat­ing chro­mo­so­mal genes.

CRISPRs may have a num­ber of pos­si­ble ap­pli­ca­tions, in­clud­ing bac­te­r­ial strain typ­ing (spoligo­typing), an­tivi­ral ther­apy, and mi­cro­bial gene si­lenc­ing. The words small in­ter­fer­ing RNAs (siR­NAs) are be­ing heard through­out the mi­cro­bial lands.