Evo­lu­tion? Who Needs It...

...Now That We Have CRISPR/Cas9s?

by Mar­cia Stone

By re­plac­ing un­wanted genes with those more to our lik­ing in germline cells, CRISPR/Cas9 en­ables us to take charge of our own des­tiny.

As re­ported on Small Things Con­sid­ered in Sep­tem­ber 12, 2015, the Vo­gons in Dou­glas Adams' Hitch­hik­ers Guide to the Galaxy con­sider evo­lu­tion a waste of time and fix every­thing they think needs fix­ing with surgery. Now hu­mans can do al­most the same thing, al­beit on a much smaller scale, with tiny mol­e­c­u­lar scis­sors called "Cas9s" car­ried to their tar­gets by DNA-seek­ing RNAs known as "CRISPRs."

Fig­ure 1. Prostet­nic Vo­gon Jeltz. Source

CRISPR/Cas9s, short for "clus­tered regu­larly inter­spaced palin­dromic repeats," and the "CRISPR-asso­ci­ated pro­tein, known as "Cas," are used by Ar­chaea and Bac­te­ria to thwart phage- and plas­mid-in­fec­tions by cut­ting apart their DNAs and sav­ing the bits to help them quickly rec­og­nize any fu­ture in­va­sion at­tempts by the same preda­tors. This an­cient ex­am­ple of adap­tive im­mu­nity has re­cently been bor­rowed from Strep­to­coc­cus pyo­genes SF370 and en­gi­neered by hu­mans to "help cure dis­eases in which genes are causal or com­plicit," says Daniel Kevles cur­rently at NYU Law School and Co­lum­bia Uni­ver­sity in New York City.

CRISPR/Cas9 achieves its ef­fects by tar­get­ing alien DNA and cleav­ing it in both so­matic cells, which are not passed down from gen­er­a­tion to gen­er­a­tion, and in germline cells, that are. The sys­tem can also re­place aber­rant genes with more nor­mal ones, if that does not hap­pen nat­u­rally.

The race is on to bring CRISPR/Cas9s into clin­i­cal use; for ex­am­ple, Jen­nifer Doudna at the Uni­ver­sity of Cal­i­for­nia, Berke­ley and col­leagues are en­gi­neer­ing ver­sions that treat Hunt­ing­ton dis­ease and Down syn­drome. At the Broad In­sti­tute of MIT and Har­vard, Feng Zhang and col­leagues are de­sign­ing CRISPR/Cas9s that tar­get genes in­volved in men­tal health dis­or­ders and can­cers. They also share their reagents widely to help oth­ers de­velop use­ful ther­a­pies.

In a first-of-its-kind in­ves­ti­ga­tion re­ported last year, the dele­tion of a mu­tated exon 23 by CRISPR/Cas9 in the mdx mouse model of Duchenne mus­cu­lar dy­s­tro­phy (DMD) en­abled ex­pres­sion of a mod­i­fied dy­s­trophin gene that re­sulted in the par­tial re­cov­ery of dy­s­trophin pro­tein in heart and skele­tal mus­cle with a con­se­quent im­prove­ment in mus­cle force. "In con­trast to exon-skip­ping strate­gies, genome edit­ing can cor­rect dis­ease-caus­ing ge­netic mu­ta­tions," says Charles Gers­bach from Duke Uni­ver­sity, Durham N.C. who led a team of sci­en­tists from around the world, in­clud­ing the Zhang group, on this project. The work es­tab­lishes CRISPR/Cas9 as a po­ten­tial ther­apy for DMD which cur­rently sen­tences ap­prox­i­mately one of every 5,000 new­born males to a pro­gres­sively dis­abling pre­ma­ture death. Im­por­tantly, the edit­ing done here only af­fects mus­cle cells and is not in­her­ited by fu­ture gen­er­a­tions.

In con­trast, us­ing CRISPR tech­nol­ogy to med­dle with germline cells raises a num­ber of eth­i­cal con­sid­er­a­tions. For ex­am­ple, it en­ables the cre­ation of "de­signer ba­bies" and even en­tirely new kinds of peo­ple. Thus a re­port of CRISPR edit­ing in hu­man em­bryos last April in the jour­nal Pro­tein & Cell caused much con­tro­versy and prompted a Sum­mit on Hu­man Gene Edit­ing con­vened by the na­tional sci­en­tific acad­e­mies of the U.S., the U.K. and China that took place in Wash­ing­ton, D.C. this past De­cem­ber. This re­search which con­firmed sus­pi­cions that such ex­per­i­ments were go­ing on was led by Jun­jiu Huang at Sun Yat-sen Uni­ver­sity in Guangzhou.

Fig­ure 2. The prin­ci­ple of CRISPR/­Cas9-me­di­ated gene dis­rup­tion. A sin­gle guide RNA (sgRNA), con­sist­ing of a cr­RNA se­quence that is spe­cific to the DNA tar­get, and a tracr­RNA se­quence that in­ter­acts with the Cas9 pro­tein (1), binds to a re­com­bi­nant form of Cas9 pro­tein that has DNA en­donu­cle­ase ac­tiv­ity (2). The re­sult­ing com­plex will cause tar­get-spe­cific dou­ble-stranded DNA cleav­age (3). The cleav­age site will be re­paired by the non-ho­mol­o­gous end join­ing (NHEJ) DNA re­pair path­way, an er­ror-prone process that may re­sult in insertions/deletions (IN­DELs) that may dis­rupt gene func­tion. Source

No­tably, the Huang group used only non-vi­able polysper­mic em­bryos, which have an ex­tra set of chro­mo­somes and die nat­u­rally af­ter go­ing through the first stages of de­vel­op­ment. They tested the abil­ity of CRISPR/Cas9 to cleave the en­doge­nous b‑globin gene (HBB) in these fa­tally de­fec­tive em­bryos us­ing the tools de­vel­oped by Zhang and col­leagues; the mu­tated form of HBB causes b‑thalassemia.

CRISPR/Cas9 suc­cess­fully cleaved the tar­geted b‑globin gene in­di­cat­ing it could even­tu­ally be a use­ful ther­apy for b‑thalassaemia. It also con­firmed that polysper­mic em­bryos, which are usu­ally dis­carded by fer­til­ity clin­ics, are a good al­ter­na­tive to vi­able hu­man em­bryos. That's the good news. Un­for­tu­nately the ef­fi­ciency of HBB re­pair was low, off-tar­get cleav­age high as were un­ex­pected mu­ta­tions. Huang and col­leagues there­fore call for fur­ther im­prove­ment in the fi­delity and speci­ficity of the CRISPR/Cas9 sys­tem as a pre­req­ui­site to clin­i­cal ap­pli­ca­tion.

The ex­pert con­sen­sus of the al­most 500 par­tic­i­pants from a va­ri­ety of dif­fer­ent fields at­tend­ing the De­cem­ber Sum­mit was that even though CRISPRs of­fer the abil­ity to erad­i­cate most, or even all, ge­netic dis­eases, no one should be rush­ing into it.

Fig­ure 3. Eight-cell hu­man em­bryo, at 3 days. Source

This is a course chal­lenged by Prostet­nic Vo­gon Jeltz who points to the ex­ten­sive ex­pe­ri­ence his peo­ple have had mak­ing their own changes as needed and how much time they've saved by "not wait­ing for na­ture to get around to mak­ing up­grades."

How­ever, in re­sponse J. Jef­frey Mor­ris says "Hu­man at­tempts to 'in­tel­li­gently de­sign' ecosys­tems doesn't leave us with a lot of con­fi­dence that we un­der­stand nature's com­plex web of in­ter­ac­tions well enough to ma­nip­u­late them on a large scale with­out caus­ing dis­as­ter: think kudzu in the [our] South and rab­bits in Aus­tralia."

"The hu­man genome is a prod­uct of evo­lu­tion si­mul­ta­ne­ously op­ti­miz­ing hun­dreds of phe­no­types ¾ if we try to al­ter one or two of those phe­no­types we think are im­por­tant, it's hard to pre­dict what ef­fect that will have on all those other phe­no­types. My in­tu­ition tells me that evo­lu­tion is way smarter than we are and we tin­ker with it at our peril," cau­tions Mor­ris, an evo­lu­tion­ist at the Uni­ver­sity at the Uni­ver­sity of Al­abama in Birm­ing­ham.

 

Sug­gested read­ing

Adams, Dou­glas (1983). Hitch­hik­ers Guide to the Galaxy. Har­mony Books, New York. ISBN 0–330-25864–8

Doudna, J.A. and Car­pen­tier, E. (2014) The new fron­tier of genome en­gi­neer­ing with CRISPR-Cas9. Science;346: 1077- DOI: 10.1126/science.1258096

Kevles, D.J. (2015) If you could de­sign your baby's genes, would you? Politico; pp.1–8.

Laing, P. et al. (2015) CRISPR/­Cas9-me­di­ated gene edit­ing in hu­man tripronu­clear zy­gotes. Pro­tein & Cell (5):363–372. DOI 10.1007/s13238-015‑0153‑5

Mor­ris JJ, Pa­poulis SE, and Lenski RE.(2014) "Co­ex­is­tence of Evolv­ing Bac­te­ria Sta­bi­lized by a Shared Black Queen Func­tion," Evo­lu­tion 68; 2960–71.

Mor­ris JJ, Lenski RE, and Zinser ER, (2012):  "The Black Queen Hy­poth­e­sis: Evo­lu­tion of De­pen­den­cies through Adap­tive Gene Loss," mBio 3 e00036-00012.

Nel­son, C.E.et al. Sci­ence 19.1126/science.aad5143 (205).

 

 Mar­cia Stone is a sci­ence writer based in New York City and fre­quent con­trib­u­tor to the ASM's Mi­crobe mag­a­zine.