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Crispr – gene editing
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CRISPR gene editing: revolution or evolution?

Erika Brunet
Erika Brunet
Inserm Research Director at Institut Imagine

In 2020, the Nobel Prize in Chem­istry was award­ed to Emmanuelle Char­p­en­tier and Jen­nifer Doud­na for a tech­nique they had invent­ed only 8 years before. Known as CRISPR/Cas9 – or “mol­e­c­u­lar scis­sors” – they first pre­sent­ed the method to the world when they pub­lished their paper in pres­ti­gious jour­nal, Sci­ence, in 2012. Since then, the tech­nique has been tout­ed as a rev­o­lu­tion in the world of mol­e­c­u­lar biol­o­gy and beyond.

INSERM researcher Dr. Eri­ka Brunet at Insti­tut Imag­ine, in the field cel­lu­lar and mol­e­c­u­lar biol­o­gy, uses the tech­nique reg­u­lar­ly in her lab­o­ra­to­ry. She says that whilst CRISPR/Cas9 has been cit­ed as a rev­o­lu­tion, pre­vi­ous tech­niques shouldn’t be for­got­ten. For her, it could not have hap­pened had oth­er researchers not paved the way.

As a sci­en­tist in the field of can­cer research, how has CRISPR-Cas9 trans­formed biology?

Eri­ka Brunet. CRISPR-Cas9 is a high­ly effec­tive tech­nique for cut­ting DNA at a pre­cise loca­tion, allow­ing the user to replace the sequence cut out with anoth­er one; a fun­da­men­tal basic of ‘gene edit­ing’. Whilst this is not the first method of DNA alter­ation that exists, it is a high­ly effec­tive, “easy to use” and flex­i­ble method. As such, it opens the door to many appli­ca­tions – most notably gene ther­a­pies, in which we could replace defec­tive genes in patients to treat them for cer­tain diseases.

There are also many research appli­ca­tions too, though. In my field, can­cerol­o­gy, we use CRISPR/Cas9 to seek new ther­a­peu­tic tar­gets. We cut out genes in cells to re-trace the steps of tumour growth. The tool its high­ly flex­i­ble, all that is need­ed is to order a desired RNA sequence or to make it – which is a sur­pris­ing­ly easy thing for a biol­o­gist to do!

For me, how­ev­er, it is impor­tant to point out that CRISPR/Cas9 would not exist had it not been for the many years of research that had paved the way before it. Yes, the CRIP­SR-Cas9 that won the Nobel Prize is the ‘new gen­er­a­tion’ of mol­e­c­u­lar scis­sors. But it is a tech­nique that is more of an evo­lu­tion in mol­e­c­u­lar tech­nol­o­gy rather than a com­plete­ly new idea. We don’t hear enough about that.

CRISPR is a fan­tas­tic tech­nique as it works “in the blink of an eye” – the design of CRISPR/cas9 sys­tem for one DNA tar­get only takes a few days. Also, before when we want­ed to cut DNA in a pre­cise loca­tion, we would be suc­cess­ful in 1 cell out of very ~1 mil­lion test­ed. With CRISPR, we are fre­quent­ly suc­cess­ful in 1 cell per 100 – so the jump is enor­mous.

Pre­vi­ous­ly, when we want­ed to cut the DNA at a spe­cif­ic point, we suc­ceed­ed in about one cell in a million.

In your research, you seek to bet­ter under­stand tumours. How do you use CRISPR-Cas9?

In my lab­o­ra­to­ry we study tumoral cells, specif­i­cal­ly the process of how a nor­mal cell becomes can­cer­ous. Many can­cers such as leukaemia and lym­phomas devel­op because of an acci­den­tal genet­ic alter­ation in a process called ‘genom­ic translo­ca­tion’. It hap­pens when two chro­mo­somes in a cell crossover and swap a long piece of their DNA with one anoth­er. Most of the time when this hap­pens the cell will “cope” with this exchange of chro­mo­some seg­ment as it does not occur on an impor­tant gene sequence. But on some occa­sions a new ‘can­cer’ gene called onco­gene will be formed that will make the cells to “trans­form” and grow chaot­i­cal­ly, even­tu­al­ly lead­ing to a tumour. 

We can use CRISPR/Cas9 to study how this process works, from the moment that a nor­mal cell acquires the ‘genom­ic translo­ca­tion’. To do so, we sim­ply cut the DNA of a cell of ori­gin of the dis­ease (a blood cell for exam­ple), to recre­ate the new ‘can­cer’ gene thus turn­ing the healthy cell into a can­cer­ous one. These can­cer cells repli­cate uncon­trol­lably, grow­ing into a tumour, which we then put into a mouse for study. The impor­tant aspect is that, under exper­i­men­tal con­di­tions, we can repli­cate the very same series of DNA ‘events’ that would hap­pen in real-life and dis­sect the tumour process from the ori­gin. This allows us to bet­ter under­stand how each DNA alter­ation of a cell can even­tu­al­ly become leukaemia, lym­phoma, or any oth­er can­cer. Ulti­mate­ly, we can iden­ti­fy new tumour mark­ers and ther­a­peu­tic targets.

What advan­tages does the CRISPR-Cas9 tech­nique have in your field?

We work on spe­cif­ic can­cers such as Ewing’s sar­co­ma, a bone can­cer which main­ly affects chil­dren and ado­les­cents. Cur­rent­ly in most cas­es, the prog­nos­tic is very bad – the can­cer metas­ta­sis­es (mean­ing it spreads to the rest of the body) in as many as 30% of patients. Whilst we have a hard time effec­tive­ly treat­ing Ewing’s sar­co­ma, we do know a fair amount about its ori­gins. In ~90% of cas­es, the dis­ease stems from a genom­ic translo­ca­tion that hap­pens when chro­mo­somes 11 and 22 crossover acci­den­tal­ly. As such, we use CRISPR-Cas9 re-cre­ate the error by pre­cise­ly cut­ting chro­mo­somes 11 and 22 that can occur in cells at the ori­gin of Ewing’s sar­co­ma tumours. Using com­bi­na­tions of dif­fer­ent patient muta­tions induced by CRISPR/Cas9, we recent­ly obtained a unique mod­el of Ewing sar­co­ma­ge­n­e­sis that should be valu­able for the sci­en­tif­ic com­mu­ni­ty work­ing on this par­tic­u­lar­ly aggres­sive pae­di­atric cancer.

What exist­ed before CRISPR-Cas9?

At the very begin­ning, when I start­ed my research in cell biol­o­gy, I would use short pieces of DNA attached to chem­i­cals to alter DNA. They were very niche and could be used to tar­get mul­ti­ple short DNA sequences. Next, there were what we called meganu­cle­as­es and zinc-fin­ger nucle­as­es, which were a step up but still dif­fi­cult to design and engi­neer – so not acces­si­ble to every­one. Impor­tant­ly, how­ev­er, using this ‘first gen­er­a­tion’ of DNA nucle­as­es we saw real achieve­ments based on these tech­niques; zinc-fin­ger nucle­as­es are used to reach clin­i­cal tri­als for cures to HIV infec­tion. Then, in 2010 we saw the arrival of TAL­ENs. They were much eas­i­er to han­dle, being assem­bled in the lab­o­ra­to­ry in under three weeks with a sim­pli­fied code to recog­nise each base pair of DNA. This real­ly put nucle­as­es on the map for wide­spread use. But two years lat­er CRISPR/Cas9 arrived, knock­ing TAL­ENs off their throne. There­fore, it could be said that oth­ers had done a lot of the leg work devel­op­ing tech­niques and get­ting gene edit­ing out there for numer­ous types of cells in dif­fer­ent species, paving the way for CRISPR.

Interview by James Bowers

Contributors

Erika Brunet

Erika Brunet

Inserm Research Director at Institut Imagine

Erika Brunet works on cancer biology to understand how DNA alterations induce the appearance of cancers. Using genome editing methods like CRISPR/Cas9, she deciphers how normal cells become cancerous in order to identify new therapeutic targets. She works as research director at the INSERM, in the Genome Dynamics in the Immune System lab at the Imagine Institute, Paris.