Why CRISPR Doesn't Work in E. coli

by Elio

We re­ceived this query:

»I en­joyed the ar­ti­cle on your blog 'Six Ques­tions About CRISPRs' by Merry Youle. I am an ex-lamb­dol­o­gist, hav­ing quit phage lambda in the early 70s and moved to GM-plants. There is one thing about CRISPR that I do not un­der­stand: Why did lamb­dol­o­gists not find CRISPR?
They found phage re­sis­tance and lysogeny and re­stric­tion sys­tems but not CRISPR. I sup­pose that they were happy that phage re­sis­tance was MalB and never looked any fur­ther, though. 27 lambda-re­sis­tant mu­ta­tions were mapped by Thirion & Hof­nung and they all mapped in malB/malT. I hope that you or Merry Youle can help me?
Best re­gards«
John Davi­son

John Davison

John Davi­son, French In­sti­tute for Agri­cul­tural Re­search, Ver­sailles

We turned to Udi Qim­ron, a mi­cro­bial ge­neti­cist and ex­pert on CRISPRs for help. He re­sponded:

Udi Qimron

Udi Qim­ron in his lab at the De­part­ment of Hu­man Mi­cro­bi­ol­ogy of Tel Aviv Uni­ver­sity

»The an­swer to why lam­dol­o­gists did not iden­tify CRISPR-Cas sys­tems is ba­si­cally be­cause they could not. The CRISPR-Cas sys­tem is in­ac­tive in E. coli  due to the H‑NS reg­u­la­tor (en­coded by the hns gene). Both the adap­ta­tion and in­ter­fer­ence steps are re­pressed in the pres­ence of H‑NS. There­fore, the sys­tem does not ac­quire new spac­ers nat­u­rally (see here) and even if it did, these spac­ers would not pro­tect, as the rel­e­vant in­ter­fer­ence pro­teins are not ex­pressed. Re­sis­tance against phages in E. coli  will thus be much more easy to achieve by other means, es­pe­cially by ad­sorp­tion mu­ta­tions (e.g. in LamB). Thus, even now, there is no wild type E. coli that one can "train" to pro­tect from phages us­ing the CRISPR-Cas. Us­ing an hns− mu­tant, one could, over the course of two weeks, gain pro­tec­tion against plas­mids (e.g. see here), and po­ten­tially against per­sis­tent phages such as M13. How­ever, the prob­a­bil­ity that one would carry out such ex­per­i­ments over such a long pe­riod, in the spe­cific mu­tant, with­out prior knowl­edge of the CRISPR-Cas is un­likely.
In other bac­te­ria, such as in Strep­to­coc­cus ther­mophilus, the sys­tem ac­tu­ally serves as the dom­i­nant de­fense sys­tem (with nearly all phage re­sis­tance oc­cur­ring due to this sys­tem) and were some "lam­dol­o­gists" stud­ied this bac­terium, they would eas­ily iden­tify it, as did Bar­ran­gou et al. in 2007.

 

Fron­tispiece: A bunch of Lambda phages. Source

 

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