Six Ques­tions About CRISPRs

by Merry Youle

Phage pre­da­tion on bac­te­ria is in­tense, but bac­te­ria are not de­fense­less sit­ting ducks. They make use of a reper­toire of di­verse strate­gies to stay even with even the wil­i­est of phages. First line de­fenses are those that block phage en­try at the door. Of­ten these in­volve mod­i­fy­ing a sur­face com­po­nent that is used by the phages as a recog­ni­tion, at­tach­ment, or en­try site. Such changes typ­i­cally carry fit­ness costs, by im­pact­ing es­sen­tial trans­porters for ex­am­ple; as a re­sult, phage sen­si­tive strains of­ten out­com­pete the phage-re­sis­tant mu­tants when there are no phages around. Also, when re­sis­tance is gained by mod­i­fy­ing the bacterium's sur­face LPS or O‑antigen, ac­quir­ing re­sis­tance to one phage can mean the loss of re­sis­tance to an­other.

CRISPRs, on the other hand, are a sec­ond line phage de­fense, one that swings into ac­tion af­ter the phage has suc­cess­fully at­tached and in­jected its genome into the cell. Al­though it may be risky let­ting the wolf in the door, a suc­cess­ful CRISPR de­fense of­fers sev­eral ad­van­tages. For one, the bac­terium may get a nu­cleotide lunch; for an­other, CRISPRs can de­fend against plas­mids, too. And as we'll see, this sys­tem al­lows for re­sis­tance to mul­ti­ple phages with­out ap­par­ent fit­ness costs. So far CRISPRs have been found in ap­prox­i­mately 40% of the se­quenced bac­te­r­ial genomes, but that num­ber may be an un­der­es­ti­mate as many of the se­quenced strains, hav­ing been main­tained in phage-free cul­ture for a long time, may have lost their CRISPRs.

What is a CRISPR?

For the ba­sic story about these Clus­tered Regu­larly InterSpersed Palin­dromic Repeats, as known three years ago, check out our ear­lier post and re­fer to the fig­ure leg­end be­low. For ex­cel­lent re­cent re­view ar­ti­cles, click here, here, and here.

A CRISPR lo­cus in a bac­te­r­ial chro­mo­some. The lo­cus in­cludes an ar­ray of al­ter­nat­ing spac­ers and palin­dromic di­rect re­peats. The iden­ti­cal re­peats range be­tween 21 and 47 bp in dif­fer­ent loci; the spac­ers are of con­stant length but are hy­per­vari­able in se­quence, their se­quences hav­ing been de­rived from pre­vi­ously en­coun­tered DNA phages or plas­mids. The en­tire ar­ray is tran­scribed as a sin­gle mRNA un­der the di­rec­tion of a pro­moter lo­cated in the leader se­quence. Other CRISPR-as­so­ci­ated genes (CAS genes) en­code the CAS pro­teins that add new spacer-re­peat pairs, process the CRISPR tran­script, and cleave the rec­og­nized for­eign DNA. Source

How do CRISPRs work?

An im­por­tant clue came from ob­ser­va­tion of bac­te­r­ial mu­tants that have ac­quired phage re­sis­tance. Among them were some that have added a new re­peat-spacer pair at the leader end of the ar­ray. Each of these new spacer se­quences matches some sec­tion of the in­fect­ing phage genome (called the pro­to­spacer). How a bac­terium does this is not yet known; the re­searchers who fig­ure this out will surely make a big splash.

Since those added spac­ers are nec­es­sary for the newly ac­quired phage re­sis­tance, the CRISPR sys­tem is a bla­tant smok­ing gun. But just how do CRISPRs in­ter­fere with phage in­fec­tion? As noted in one of the 2010 re­views: The mech­a­nism by which CRISPR pro­vides re­sis­tance against for­eign ge­netic el­e­ments is not fully char­ac­ter­ized. Some mech­a­nisms have been ruled out: CRISPR de­fense does not block phage ad­sorp­tion or DNA in­jec­tion, does not in­volve a re­stric­tion-mod­i­fi­ca­tion sys­tem, and is not an abortive in­fec­tion mech­a­nism. Most likely, the degra­da­tion of the tar­geted nu­cleic acid by a CRISPR en­donu­cle­ase is the key.

The CRISPR de­fense mech­a­nism. From this source, slightly mod­i­fied.

Here are some of the known de­tails. The en­tire CRISPR ar­ray is tran­scribed con­sti­tu­tively as a sin­gle, long RNA that is then cut at a spe­cific site in each re­peat to yield the ma­ture CRISPR RNAs (cr­RNAs). Each cr­RNA con­tains one en­tire spacer se­quence plus rec­og­niz­able "han­dles" pro­vided by short 3' and 5' flank­ing re­gions de­rived from the ad­ja­cent re­peats. The pre­cise cut­ting is done by a com­plex of the CAS pro­teins called Cas­cade. Some of these pro­teins re­main bound to the cr­RNAs to form the ac­tive de­fense agents. The spacer se­quence pro­vided by the cr­RNA is thought to rec­og­nize and guide the com­plex to the spe­cific tar­get se­quence; then one (or more) of the pro­teins with nu­cle­ase ac­tiv­ity cuts the in­vad­ing nu­cleic acid. Since all the spac­ers are con­sti­tu­tively tran­scribed and processed into ac­tive de­fense agents, the host is con­tin­u­ously on guard against all the cor­re­spond­ing phages and plas­mids.

When this story first came to light, piece by piece, there were spec­u­la­tions that the CRISPR mech­a­nism might be anal­o­gous to RNA in­ter­fer­ence in eu­kary­otes. In­deed, there are anal­o­gous steps in­volved in the for­ma­tion of the ac­tive de­fense com­plexes in both sys­tems. How­ever, in bac­te­ria, the tar­get is for­eign DNA, not RNA. Ev­i­dence? Spac­ers com­ple­men­tary to ei­ther the cod­ing or non-cod­ing strand of phage λ con­fer re­sis­tance. Other ex­per­i­ments demon­strat­ing this used the CRISPR lo­cus of a clin­i­cal iso­late of Staphy­lo­coc­cus epi­der­midis and a plas­mid with an in­tron-con­tain­ing gene. Here the re­searchers ex­per­i­men­tally in­tro­duced spac­ers that matched ei­ther the plasmid's gene se­quence (in­clud­ing the in­tron) or the cor­re­spond­ing spliced mRNA se­quence (lack­ing the in­tron). Only spac­ers against the in­tron-con­tain­ing DNA se­quence were ef­fec­tive.

That said, I have to men­tion that the Ar­chaea, as usual, do it their own way. CRISPR loci have been found in vir­tu­ally all se­quenced ar­chaeal genomes, but they tar­get for­eign RNA, not DNA. (That opens to door to all sorts of evo­lu­tion­ary spec­u­la­tions!)

How do phages evade CRISPRs?

The bac­te­r­ial CRISPR de­fense is very finicky and will cleave for­eign DNA only if it con­tains a se­quence that ex­actly matches a spacer. Thus phage eva­sion is only a sin­gle base change away. That phage do in­deed es­cape this way has been shown by chal­leng­ing cul­tures with a phage for which they al­ready have a spacer. One finds a small num­ber of phages that are able to suc­cess­fully mul­ti­ply. A close look at the genome se­quence of these phages typ­i­cally re­veals a sin­gle nu­cleotide change in the pro­to­spacer. (The ar­chaeal sys­tem is less ex­act­ing, re­quir­ing more than one nu­cleotide change in that re­gion for es­cape.)

Al­though pro­to­spac­ers are found on both cod­ing and non-cod­ing strands through­out the phage genome, their lo­ca­tions are not en­tirely ran­dom. They are al­ways just a few base pairs from a short mo­tif that is rec­og­nized by the CRISPR sys­tem. Phages with­out that mo­tif in their genome are im­mune. An­other eva­sion trick used by viruses and other mo­bile el­e­ments is to in­sert them­selves in one of the CAS genes or oth­er­wise in­ter­fere with the op­er­a­tion of the CRISPR. This might be one of the fac­tors that make it worth­while for hosts to carry more than one CRISPR lo­cus. (The cur­rent record is 18 loci, ac­count­ing for 1% of the genome in the ar­chaeon Methanocal­do­coc­cus jan­naschii.)

Do mo­bile el­e­ments also use CRISPRs?

Since all the genes needed for a CRISPR de­fense sys­tem are clus­tered to­gether, whole CRISPR loci are good can­di­dates for swap­ping around by hor­i­zon­tal gene trans­fer. Al­ready CRISPR loci have been found on at least 10 megaplas­mids. This has a cou­ple of im­pli­ca­tions. For one, megaplas­mids may be the ve­hi­cle by which en­tire loci travel be­tween bac­te­r­ial lin­eages. Phy­lo­ge­netic ev­i­dence clearly shows that such trans­fers have hap­pened. These plas­mids might also be us­ing CRISPRs to com­pete with other plas­mids for their bac­te­r­ial hosts. In­deed, some CRISPR-bear­ing plas­mids have spac­ers that tar­get the slightly dif­fer­ent CRISPR genes on other, po­ten­tially com­pet­ing plas­mids. Viruses them­selves may also use CRISPRs. Two Clostrid­ium dif­fi­cile prophages carry CRISPR loci — pos­si­bly a mech­a­nism to limit su­per­in­fec­tion by other phages.

This plot of the abun­dance dis­tri­b­u­tion of CRISPR spac­ers re­flects the his­tory of hor­i­zon­tal trans­fer of the CRISPR lo­cus be­tween the Lep­tospir­il­lum strains sam­pled from two Rich­mond Mine lo­ca­tions. The num­ber of oc­cur­rences of each spacer was counted at each lo­ca­tion and plot­ted. A and B = the two lo­ca­tions. Red = spac­ers found at both lo­ca­tions; blue and green = spac­ers present in only lo­ca­tion A or B. The most abun­dant spac­ers at both lo­ca­tions are the older spac­ers that were present prior to the hor­i­zon­tal trans­fer be­tween the two strains; the less abun­dant spac­ers were ac­quired more re­cently, af­ter the trans­fer, and thus they are found in fewer in­di­vid­u­als and in only one lo­ca­tion. Other analy­ses show that they are lo­cated at the leader end of the ar­ray. Source

What can we learn from CRISPR ar­rays?

A CRISPR ar­ray is akin to a fos­sil record that re­veals the his­tory of pre­vi­ous vi­ral chal­lenges. New spac­ers are added at the leader end of the ar­ray, the old­est spac­ers are found at the trail­ing end. Mech­a­nisms for elim­i­nat­ing old repeat/spacer pairs limit the growth of the ar­ray and also can pro­duce gaps in the his­tory. Nev­er­the­less, close scrutiny of an ar­ray can tell us much about the bacterium's past.

The Ban­field lab used this record to un­cover the his­tory of closely-re­lated strains of Lep­tospir­il­lum group II sam­pled from two acid mine drainage lo­ca­tions in the Rich­mond Mine. (Click here for Elio's ear­lier post about the ar­chaeal in­hab­i­tants of this mine.) These are ex­treme en­vi­ron­ments: pH ~1, tem­per­a­ture ~40 °C. The story told by their CRISPR ar­rays is that the strain at one lo­ca­tion had ac­quired their CRISPR lo­cus from the other by HGT. Ev­i­dence? Over­all, their genomes show only 94% iden­tity, but the 5 kb re­gion that spans their CRISPR loci is iden­ti­cal (ex­cept for the spac­ers). Based on more de­tailed analy­sis of the strain from one lo­ca­tion, the re­searchers note that the pop­u­la­tion ap­pears to be nearly clonal, ex­cept for their rapidly-evolv­ing spac­ers. In­deed, the spac­ers are so het­ero­ge­neous that no two sam­pled in­di­vid­u­als in that pop­u­la­tion share the ex­act same com­ple­ment.

How might CRISPRs af­fect bac­te­r­ial evo­lu­tion?

CRISPRs re­duce the fre­quency of hor­i­zon­tal gene trans­fer. By de­fend­ing against in­com­ing phage or plas­mid DNA, they erect a bar­rier against two of the main mech­a­nisms of HGT: trans­duc­tion via viruses and con­ju­ga­tion via plas­mids. Where there are many CRISPR loci, plas­mids are less com­mon. Strep­to­coc­cus ther­mophilus, for ex­am­ple, has 12 CRISPR loci and few plas­mids. Since plas­mids of­ten carry an­tibi­otic re­sis­tance genes, some peo­ple think that CRISPRs might be help­ful al­lies in our at­tempts to re­duce the spread of an­tibi­otic re­sis­tance. From the bac­te­r­ial per­spec­tive, though, plas­mids can be use­ful sources of spe­cial­iza­tion genes — genes for an­tibi­otic and heavy metal re­sis­tance, fer­men­ta­tion of sug­ars and aro­matic com­pounds, toxin pro­duc­tion, and re­stric­tion-mod­i­fi­ca­tion sys­tems, among oth­ers.

A CRISPR de­fense can also make it dif­fi­cult for a tem­per­ate phage to in­sert as a prophage. In some bac­te­r­ial groups, there is a neg­a­tive cor­re­la­tion be­tween the num­ber of prophages and the num­ber of CRISPR ar­rays. Ex­clud­ing po­ten­tial prophage can be an­other cost to the host since prophages can pro­vide use­ful genes (e.g., vir­u­lence genes such as those for the diph­the­ria and cholera tox­ins). Thus, when eval­u­at­ing the fit­ness costs of CRISPRs, it may be nec­es­sary to fac­tor in these long-term eco­log­i­cal and evo­lu­tion­ary costs, as well.

So, when shop­ping for an anti-phage de­fense, which is the best buy — a first line de­fense or a CRISPR lo­cus? Or both?

Ref­er­ences

Hor­vath P, Bar­ran­gou R. (2010). CRISPR/Cas, the Im­mune Sys­tem of Bac­te­ria and Ar­chaea Sci­ence, 327 (5962), 167−170. DOI 10.1126/science.1179555

Karginov FV, Han­non GJ. (2010). The CRISPR sys­tem: small RNA-guided de­fense in bac­te­ria and ar­chaea. Mol­e­c­u­lar cell, 37 (1), 7−19. PMID 20129051

Mar­raf­fini LA, Son­theimer EJ. (2010). CRISPR in­ter­fer­ence: RNA-di­rected adap­tive im­mu­nity in bac­te­ria and ar­chaea. Na­ture re­views. Ge­net­ics, 11 (3), 181−190. PMID 20125085