Modifying DNA: applications of the CRISPR discovery

As the reg­u­la­tion of genet­ic mod­i­fi­ca­tion is chang­ing in Euro­pean Par­lia­ment¹, the CRISPR-Cas9 gene-edit­ing tech­nol­o­gy divides opin­ion. A source of con­cern for some, full of promise for oth­ers, let’s take a look at how it works, the pos­si­bil­i­ties it opens up, its lim­its and the ques­tions it raises.

CRISPRs (Clus­tered reg­u­lar­ly inter­spaced short palin­dromic repeats) are DNA sequences found in the genomes of many prokary­otes, both bac­te­ria and archaea. First iden­ti­fied in Escherichia coli in 1987², they have a char­ac­ter­is­tic struc­ture: short, con­served repeats are sep­a­rat­ed by oth­er short, vari­able sequences known as spac­ers. It took a great deal of research over 20 years³ to under­stand that these are defence ele­ments against virus­es⁴.

Indeed, spac­er sequences are por­tions of viral genomes, inte­grat­ed by prokary­otes into their own genome. CRISPR DNA is tran­scribed into RNA, which in turn is cut to form small guide RNAs called crRNAs (CRISPR RNAs), derived from each of the spac­ers and there­fore sim­i­lar to viral sequences. When a virus cor­re­spond­ing to one of these spac­ers infects the cell, the asso­ci­at­ed crRNA binds to its genome. The struc­ture thus formed is then tar­get­ed by an enzyme of the Cas fam­i­ly (for CRISPR-asso­ci­at­ed), capa­ble of cut­ting the viral DNA, thus putting an end to the infection.

The CRISPR-Cas sys­tem there­fore works in three stages: expres­sion of crRNAs, recog­ni­tion of the viral genome by a crRNA, and cut­ting by a Cas pro­tein, guid­ed to the right place by the crRNA. When a prokary­ote encoun­ters a new virus, it can recov­er a piece of its genome and add it to its CRISPR library: the CRISPR-Cas immune response is there­fore spe­cif­ic to each virus encoun­tered. What’s more, this mem­o­ry is trans­mit­ted hered­i­tar­i­ly, since it is stored direct­ly in the genome. If this remark­able defence mech­a­nism is now known well beyond the micro­bi­ol­o­gy com­mu­ni­ty, it’s because it has been adapt­ed to design a par­tic­u­lar­ly easy-to-use gene-edit­ing tool.

The gen­er­al idea is to use the mol­e­c­u­lar scis­sors known as Cas enzymes to cut DNA where you want it, using a cus­tom-designed crRNA to recog­nise a spe­cif­ic genet­ic sequence. Once the DNA has been cut, sev­er­al things can hap­pen: if the cell is left to repair the break, the sequence is altered in the process. But if it is pro­vid­ed with a DNA mod­el, con­tain­ing a rel­e­vant sequence (bor­dered by the same motifs as those found on either side of the break), this is used by the cell in a repair process called homol­o­gous recom­bi­na­tion, a kind of mol­e­c­u­lar copy-and-paste. The DNA mod­el sequence is inte­grat­ed into the genome at the point where the cut was made.

By cut­ting where you want to insert any DNA sequence, the CRISPR-Cas9 tech­nique makes it pos­si­ble to mod­i­fy genomes with extreme pre­ci­sion. This makes it pos­si­ble to intro­duce small muta­tions into the DNA, add new genes or, if no repair mod­el is pro­vid­ed, deac­ti­vate a gene. As it is now easy and inex­pen­sive to order cus­tomised nucle­ic acids for use as crRNA, this method of gene edit­ing is very acces­si­ble, as well as being high­ly effective.

By cut­ting where you want to insert any DNA sequence, the CRISPR-Cas9 tech­nique makes it pos­si­ble to mod­i­fy genomes with extreme precision.

In prokary­otes, there are a wide vari­ety of Cas pro­teins with dif­fer­ent func­tions and char­ac­ter­is­tics⁵. The first gene-edit­ing sys­tems were devel­oped with Cas9, which pro­duces dou­ble-strand DNA cuts. How­ev­er, sev­er­al ver­sions of this enzyme, iso­lat­ed from dif­fer­ent micro-organ­isms, recog­nise sequences of slight­ly dif­fer­ent lengths with vary­ing speci­fici­ties. Oth­er Cas enzymes are of par­tic­u­lar inter­est, notably Cas12, which func­tions even more sim­ply than Cas9. It shows promise for simul­ta­ne­ous­ly mod­i­fy­ing sev­er­al sequences, for act­ing on the epigenome [edi­tor’s note: all epi­ge­net­ic mod­i­fi­ca­tions (mod­i­fy­ing the expres­sion of a gene) in a cell] and as a DNA detec­tion sys­tem, par­tic­u­lar­ly for diag­nos­tic tests. Cas13 cuts RNA rather than DNA, mak­ing it an inter­est­ing tool for detect­ing RNA, includ­ing infec­tious RNA, and open­ing up the pos­si­bil­i­ty of act­ing on the tran­scrip­tome [edi­tor’s note: all the RNA result­ing from the tran­scrip­tion of the genome present at a giv­en moment], the result of the expres­sion of a genome, with­out mod­i­fy­ing the genome itself.

The CRISPR-Cas gene-edit­ing field is cur­rent­ly boom­ing, mobil­is­ing a large num­ber of research teams. Pio­neers Emmanuelle Char­p­en­tier and Jen­nifer Doud­na were award­ed the Nobel Prize in Chem­istry in 2020 for their devel­op­ment of gene edit­ing using CRISPR-Cas9⁶, fol­low­ing a sem­i­nal paper they pub­lished in 2012⁷. At the same time, oth­er teams pub­lished the first results of eukary­ot­ic cell genome mod­i­fi­ca­tions using CRISPR-Cas9 tech­nol­o­gy, includ­ing those led by Feng Zhang⁸ and his col­lab­o­ra­tor and for­mer men­tor, George Church⁹. These four sci­en­tists have since found­ed biotech­nol­o­gy com­pa­nies focused on gene edit­ing, and their super­vi­so­ry insti­tu­tions are wag­ing a ver­i­ta­ble patent war. Because the pos­si­bil­i­ties opened up by CRISPR-Cas tech­nol­o­gy have major finan­cial implications. 

Devel­oped just ten years ago, the CRISPR-Cas approach has already become a stan­dard in fun­da­men­tal biol­o­gy research lab­o­ra­to­ries. Its speed and acces­si­bil­i­ty make it an ide­al tool for switch­ing genes off or mod­i­fy­ing them, enabling a bet­ter under­stand­ing of their func­tions. It is now pos­si­ble to car­ry out screens on an entire genome rel­a­tive­ly eas­i­ly. But the fields of appli­ca­tion are much wider, and the boom is par­tic­u­lar­ly marked in three areas: health, agron­o­my and bioproduction.

The abil­i­ty to eas­i­ly mod­i­fy genes is par­tic­u­lar­ly promis­ing for treat­ing mono­genic dis­eases, i.e. those caused by the mal­func­tion­ing of a sin­gle gene. CRISPR-Cas9 makes it pos­si­ble to envis­age a new form of gene ther­a­py, ensur­ing the last­ing replace­ment of a mutat­ed allele by a func­tion­al ver­sion. The first CRISPR-Cas ther­a­py to be approved by the health author­i­ties at the end of 2023 con­cerns genet­ic dis­eases. Cas­gevy, pro­duced by Ver­tex and CRISPR Ther­a­peu­tics and now approved in the Unit­ed States and the Unit­ed King­dom, tar­gets forms of chron­ic anaemia caused by a muta­tion in one of the haemo­glo­bin genes. In France, this treat­ment has ben­e­fit­ed from the ear­ly access autho­ri­sa­tion exemp­tion since 18^th Jan­u­ary 2024¹⁰.

CRISPR-Cas could make it eas­i­er to treat dis­eases caused by the mal­func­tion­ing of sev­er­al genes, but it can­not over­come all the dif­fi­cul­ties posed by gene ther­a­pies. While cer­tain organs such as the blood, eye and liv­er are eas­i­ly acces­si­ble, deliv­er­ing the treat­ment to all the cells that need it with­out caus­ing side effects is still a chal­lenge. How­ev­er, the poten­tial appli­ca­tions of CRISPR-Cas in human health are not lim­it­ed to gene ther­a­pies and diag­nos­tic tools. Tri­als are under­way, for exam­ple, to try to elim­i­nate HIV from the bod­ies of infect­ed peo­ple¹¹, to com­bat malar­ia¹², to facil­i­tate organ trans­plants¹³, to devel­op new approach­es to dia­betes¹⁴ and many others.

Since their inven­tion, agri­cul­ture and live­stock breed­ing have been based on the genet­ic mod­i­fi­ca­tion of species in order to devel­op char­ac­ter­is­tics of inter­est to humans. This process, which involved cen­turies or even mil­len­nia of selec­tion and cross-breed­ing, has been turned on its head by the devel­op­ment of mol­e­c­u­lar biol­o­gy. Gain­ing a bet­ter under­stand­ing of the role of each gene and being able to mod­i­fy them – or even insert new ones – in a pre­cise and con­trolled way, instead of select­ing ran­dom muta­tions a pos­te­ri­ori, rep­re­sents a con­sid­er­able gain in effi­cien­cy. What’s more, the poten­tial appli­ca­tions are numer­ous. Adapt­ing the vari­eties cul­ti­vat­ed to heat, drought or oth­er envi­ron­men­tal fac­tors, improv­ing resis­tance to cer­tain pests (to reduce the use of pes­ti­cides), increas­ing yields to reduce the need for fer­tilis­ers, extend­ing the post-har­vest shelf life of prod­ucts to avoid wastage, improv­ing nutri­tion­al qual­i­ties¹⁵… Once again, it is impos­si­ble to draw up an exhaus­tive list.

The tar­get­ed mod­i­fi­ca­tions made pos­si­ble by CRISPR-Cas also make it pos­si­ble to envis­age new devel­op­ments such as the pro­duc­tion of nat­u­ral­ly decaf­feinat­ed cof­fee¹⁶ or aller­gen-free foods. Attempts are under­way, for exam­ple, to pro­duce gluten-free wheat¹⁷. Con­verse­ly, CRISPR-Cas can be used to devel­op new bio­pro­duc­tion sys­tems, by insert­ing the nec­es­sary genes into the genome of micro-organ­isms such as yeast, algae or bac­te­ria. Bio­fu­els, phar­ma­ceu­ti­cal com­pounds, cos­met­ics, food mol­e­cules such as vit­a­mins: the pos­si­bil­i­ties are numer­ous and the ease of use of CRISPR-Cas is also rev­o­lu­tion­is­ing syn­thet­ic biol­o­gy.

CRISPR-Cas is some­times pre­sent­ed as a kind of mag­ic wand, but this gene-edit­ing tool has its lim­its. It can be used to mod­i­fy genomes pre­cise­ly, but not to bring about just any change in the phe­no­type of an organ­ism. Com­plex bio­log­i­cal process­es involv­ing numer­ous genes can­not be eas­i­ly repro­grammed, even with CRISPR-Cas. Genes, which are essen­tial to the prop­er func­tion­ing of an organ­ism, can­not be mod­i­fied with­out harm­ful con­se­quences. Even if a gene con­fer­ring a giv­en prop­er­ty is known, it can­not be acquired by a new organ­ism using CRISPR-Cas. And there is noth­ing obvi­ous about trans­fer­ring lab­o­ra­to­ry pro­to­cols (car­ried out under con­trolled con­di­tions with often sim­pli­fied sys­tems) to real-life situations.

What’s more, while CRISPR-Cas edit­ing tech­nol­o­gy is par­tic­u­lar­ly pre­cise, it is not 100% reli­able. The appear­ance of so-called off-tar­get mod­i­fi­ca­tions, oth­er than at the lev­el of the sequence ini­tial­ly tar­get­ed, is a prob­lem for the ther­a­peu­tic use of CRISPR-Cas. Oth­er tech­ni­cal lim­i­ta­tions remain to be over­come¹⁸. The acces­si­bil­i­ty of poten­tial treat­ments based on CRISPR-Cas is also a point of con­cern. The price of Cas­gevy has been set at $2.2 mil­lion. Even if a sin­gle inter­ven­tion were suf­fi­cient to cure patients, the use of such an expen­sive ther­a­py is bound to remain restrict­ed and uneven.

While some uses of CRISPR-Cas seem promis­ing or anec­do­tal, oth­ers raise more dif­fi­cult eth­i­cal questions.

This is just one of the many eth­i­cal issues raised by CRISPR-Cas tech­nol­o­gy. Like any tool, nei­ther good nor bad in itself, it is its poten­tial uses that need to be exam­ined. Those that seem acces­si­ble in the near future, but also those that could become so in the longer term. Mod­i­fy­ing the genome of sick peo­ple to cure them of an oth­er­wise incur­able dis­ease seems promis­ing, but touch­ing the human genome is far from harm­less. A Chi­nese sci­en­tist who genet­i­cal­ly mod­i­fied sev­er­al embryos in an attempt to make them resis­tant to HIV was con­demned by both his peers and the courts: he spent three years in prison before being released in 2022. The fact remains that he was able to pro­duce sev­er­al genet­i­cal­ly mod­i­fied babies as part of a project whose method­ol­o­gy was as flawed as its ethics¹⁹.

So while some uses of CRISPR-Cas seem promis­ing or anec­do­tal, oth­ers raise more dif­fi­cult ques­tions. Is it accept­able to genet­i­cal­ly mod­i­fy plants to make them more resis­tant to drought? Is it accept­able to genet­i­cal­ly mod­i­fy cats to make them less aller­genic²⁰? Mos­qui­toes trans­mit dis­eases respon­si­ble for hun­dreds of thou­sands of deaths every year, but would it be accept­able to use a genet­ic forc­ing approach, based on CRISPR, to wipe out entire pop­u­la­tions of mos­qui­toes²¹? If you don’t answer these three ques­tions in the same way, what are the fac­tors that sway your opinion?

These are com­plex ques­tions, but giv­en the speed at which tech­no­log­i­cal tools are advanc­ing, it is becom­ing imper­a­tive to answer them col­lec­tive­ly. The G6 brings togeth­er six major Euro­pean research organ­i­sa­tions, includ­ing the CNRS. On the occa­sion of the recent debates in the Euro­pean Par­lia­ment, it pub­lished a press release call­ing for risk assess­ment to be “based on the char­ac­ter­is­tics of genet­i­cal­ly mod­i­fied organ­isms, rather than on the tech­niques used to obtain them.”²² On this basis, what reg­u­la­tions might be laid down?