From the smallest single-celled organism to the largest creatures on earth, every living thing is defined by its genes. The DNA contained in our genes acts like an instruction manual for our cells. Four building blocks called bases are strung together in precise sequences, which tell the cell how to behave and form the basis for our every trait. But with recent advancements in gene editing tools, scientists can change an organism’s fundamental features in record time. They can engineer drought-resistant crops and create apples that don’t brown. They might even prevent the spread of infectious outbreaks and develop cures for genetic diseases. CRISPR is the fastest, easiest, and cheapest of the gene editing tools responsible for this new wave of science. But where did this medical marvel come from? How does it work? And what can it do?
Od najmanjeg jednoćelijskog organizma do najvećih stvorenja na Zemlji, svako živo biće definisano je svojim genima. DNK koja se nalazi u našim genima deluje kao uputstvo za upotrebu našim ćelijama. Četiri gradivna bloka zvani baze su postavljeni zajedno u precizne nizove, koji govore ćeliji kako da se ponaša i formiraju osnovu za svaku našu osobinu. Sa skorašnjim naprecima alatki za uređivanje gena, naučnici mogu izmeniti osnovne odlike organizma u rekordnom vremenu. Mogu genetski modifikovati da usevi budu otporni na sušu i da stvore jabuke koje ne postaju braon. Možda čak i spreče širenje infektivnih bolesti i razviju lekove za genetske bolesti. Krispr je najbrža, najlakša i najjeftinija alatka za uređivanje gena koja je odgovorna za ovaj novi talas nauke. Nego, odakle se ovo medicinsko čudo pojavilo? Kako funkcioniše? I šta može da uradi?
Surprisingly, CRISPR is actually a natural process that’s long functioned as a bacterial immune system. Originally found defending single-celled bacteria and archaea against invading viruses, naturally occurring CRISPR uses two main components. The first are short snippets of repetitive DNA sequences called “clustered regularly interspaced short palindromic repeats,” or simply, CRISPRs. The second are Cas, or “CRISPR-associated” proteins which chop up DNA like molecular scissors. When a virus invades a bacterium, Cas proteins cut out a segment of the viral DNA to stitch into the bacterium’s CRISPR region, capturing a chemical snapshot of the infection. Those viral codes are then copied into short pieces of RNA. This molecule plays many roles in our cells, but in the case of CRISPR, RNA binds to a special protein called Cas9. The resulting complexes act like scouts, latching onto free-floating genetic material and searching for a match to the virus. If the virus invades again, the scout complex recognizes it immediately, and Cas9 swiftly destroys the viral DNA.
Iznenađujuće, Krispr je zapravo prirodan proces koji je dugo funkcionisao kao bakterijski imuni sistem. Prvobitno nađen kako brani jednoćelijske bakterije i arheje od virusa koji napadaju, prirodni Krispr koristi dve glavne komponente. Prva su kratki isečci DNK nizova koji se ponavljaju i zovu se „pravilno razmaknuta grupisana kratka palindromska ponavljanja”, ili jednostavno Krispr. Druga komponenta su kas proteini, ili proteini udruženi sa Krisprom koji seckaju DNK kao molekularne makaze. Kada virus napadne bakteriju, kas proteini odsecaju segment virusne DNK i ubacuju ga u deo bakterije gde je Krispr, pamteći hemijski snimak infekcije. Ti virusni kodovi se onda kopiraju u kratke delove RNK. Ovaj molekul ima više uloga u našim ćelijama, ali u slučaju Krispra, RNK se vezuje za specijalni protein koji se naziva kas 9. Dobijene celine se ponašaju kao izviđači koji se vezuju za genetski materijal koji slobodno pluta i traže spoj koji odgovara virusu. Ako virus ponovo napadne, izviđačka celina ga odmah prepoznaje i kas 9 brzo uništava virusnu DNK.
Lots of bacteria have this type of defense mechanism. But in 2012, scientists figured out how to hijack CRISPR to target not just viral DNA, but any DNA in almost any organism. With the right tools, this viral immune system becomes a precise gene-editing tool, which can alter DNA and change specific genes almost as easily as fixing a typo.
Mnogo bakterija ima ovaj tip odbrambenog sistema. Ali 2012. godine naučnici su otkrili kako da nasamare Krispr da cilja ne samo virusnu DNK, nego bilo koju DNK u skoro bilo kom organizmu. Sa pravim alatkama, ovaj virusni imuni sistem postaje precizna alatka za uređivanje gena, koja može da izmeni DNK i promeni određene gene lako kao da ispravlja slovnu grešku.
Here’s how it works in the lab: scientists design a “guide” RNA to match the gene they want to edit, and attach it to Cas9. Like the viral RNA in the CRISPR immune system, the guide RNA directs Cas9 to the target gene, and the protein’s molecular scissors snip the DNA. This is the key to CRISPR’s power: just by injecting Cas9 bound to a short piece of custom guide RNA scientists can edit practically any gene in the genome.
Evo kako funkcioniše u laboratoriji: naučnici dizajniraju RNK „vodiča” tako da odgovara genu koji žele da izmene, i vezuju ga za kas 9. Kao virusna RNK u imunom sistemu Krispra, RNK vodič usmerava kas 9 do ciljanog gena, i proteinove molekularne makaze odrezuju DNK. To je ključ Krisprove moći: samim ubrizgavanjem kas 9 proteina vezanog za kratki deo prilagođenog RNK vodiča, naučnici mogu da izmene skoro bilo koji gen u genomu.
Once the DNA is cut, the cell will try to repair it. Typically, proteins called nucleases trim the broken ends and join them back together. But this type of repair process, called nonhomologous end joining, is prone to mistakes and can lead to extra or missing bases. The resulting gene is often unusable and turned off. However, if scientists add a separate sequence of template DNA to their CRISPR cocktail, cellular proteins can perform a different DNA repair process, called homology directed repair. This template DNA is used as a blueprint to guide the rebuilding process, repairing a defective gene or even inserting a completely new one.
Kad je DNK isečena, ćelija će pokušati da je popravi. Obično, proteini zvani nukleaze dovode u red oštećene krajeve i spajaju ih ponovo. Ali ovaj tip procesa popravke, zvan spajanje nehomologih krajeva, je sklon greškama i može dovesti do dodatnih ili izgubljenih baza. Dobijeni gen je često neupotrebljiv i ugašen. Međutim, ako naučnici dodaju odvojeni niz šablonske DNK svom koktelu Krispra, ćelijski proteini mogu da izvrše drugačiji proces popravke DNK, zvan popravka usmerena na homologiju. Ovaj šablon DNK je korišćen kao plan za rukovođenje procesom ponovne izgradnje kojim se popravlja neispravan gen ili čak ubacuje potpuno novi.
The ability to fix DNA errors means that CRISPR could potentially create new treatments for diseases linked to specific genetic errors, like cystic fibrosis or sickle cell anemia. And since it’s not limited to humans, the applications are almost endless. CRISPR could create plants that yield larger fruit, mosquitoes that can’t transmit malaria, or even reprogram drug-resistant cancer cells. It’s also a powerful tool for studying the genome, allowing scientists to watch what happens when genes are turned off or changed within an organism.
Sposobnost da popravlja greške DNK znači da bi Krispr potencijalno mogao da stvori nove lekove za bolesti u vezi sa specifičnim genetskim greškama, poput cistične fibroze i srpaste anemije. Budući da nije ograničen samo na ljude, upotrebe su skoro beskrajne. Krispr bi mogao da stvori biljke koje rađaju veće voće, komarce koji ne mogu da prenesu malariju, ili čak da reprogramira ćelije raka koje su otporne na lekove. Takođe je moćna alatka za proučavanje genoma, koja dopušta naučnicima da gledaju šta se dešava kad su geni ugašeni ili promenjeni unutar organizma.
CRISPR isn’t perfect yet. It doesn’t always make just the intended changes, and since it’s difficult to predict the long-term implications of a CRISPR edit, this technology raises big ethical questions. It’s up to us to decide the best course forward as CRISPR leaves single-celled organisms behind and heads into labs, farms, hospitals, and organisms around the world.
Krispr još nije usavršen. Ne pravi uvek samo zamišljene promene, i budući da je teško predvideti dugoročne posledice uređivanja Krisprom, ova tehnologija postavlja značajna etička pitanja. Na nama je da odlučimo o najboljem pravcu dok Krispr ostavlja jednoćelijske organizme iza sebe i upućuje se u laboratorije, farme, bolnice i organizme širom sveta.