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Biology 2012

A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity

Martin Jinek, Emmanuelle Charpentier, Jennifer Doudna et al.

Reprogram a bacterial defence enzyme with a short RNA, and you can cut any gene you choose.

Choose your version
In depth · the introduction

Scientists took a defence system that bacteria use against viruses and turned it into cheap, precise “scissors” that can find and cut any chosen gene.

The idea, unpacked

Bacteria have their own immune system. When a virus attacks, they save a small piece of its DNA as a kind of mugshot, then make a matching strand of RNA that guides a cutting protein — Cas9 — to chop up that exact sequence if the virus ever returns. The guide RNA is essentially a search term, and Cas9 is the blade.

Where it came from

For years, CRISPR was a curiosity of bacterial biology. The breakthrough was realising you can write your own search term. Swap in a guide RNA that matches any gene you like — in a plant, a mouse, or a human cell — and Cas9 will travel to that precise spot in the three-billion-letter genome and make a cut. The cell then tries to repair the break, and scientists can use that moment to switch a gene off or paste in a corrected version. Older gene-editing methods meant building a complicated custom protein for every target. CRISPR just needs a new snippet of RNA — so cheap and simple that labs everywhere adopted it almost overnight, and Charpentier and Doudna shared the 2020 Nobel Prize in Chemistry.

Why it mattered

CRISPR put precise gene editing within reach of ordinary laboratories, transforming biology and medicine. It's now being used to develop treatments for inherited diseases like sickle-cell anaemia, to engineer hardier crops, and to study how genes work — which is also why it raises serious ethical questions about editing human life.

Two locks, not one

Aiming the scissors isn't quite as simple as “any address.” Cas9 first checks for a tiny three-letter pass code in the DNA — a “PAM,” the letters NGG — sitting right next to the target. No pass code, no cut, even if the guide matches perfectly. Then the guide has to pair up with the DNA letter by letter, and a mismatch in the stretch nearest the pass code is enough to stop it. Try writing a guide and watch Cas9 decide whether to cut.

A short stretch of double-stranded DNA with a 20-letter guide RNA above it. Each guide letter is coloured green where it pairs with the DNA target and red where it does not; a highlighted three-letter PAM (NGG) sits just past the target. When the PAM is present and the PAM-proximal letters all match, a pair of scissors marks a blunt cut three letters upstream of the PAM. Clicking a guide letter changes it, and toggling the PAM removes the cut.

What came next

CRISPR moved from the lab bench to the clinic with startling speed. In 2023 the first CRISPR-based therapy was approved, curing some patients of sickle-cell disease by editing their own blood cells. Newer versions can rewrite a single DNA letter without even cutting both strands. The same power is why the world is still debating where to draw the line — especially around editing embryos, whose changes would pass to every future generation.

The original document
Original source text
M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, E. Charpentier · Science 337 (2012): 816–821
The system
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids.
Mechanism
We show that in a subset of these systems, the mature crRNA … forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 … protein cleaves both strands.
Engineering a single guide
We engineered the two RNAs into a single RNA chimera and show that it directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
Cas9 can be programmed using a single engineered RNA molecule … to cleave any dsDNA sequence of interest, providing a simple two-component system for targeted genome editing.
The full paper — with the biochemical cleavage assays, the mapping of the HNH and RuvC nuclease domains to the two DNA strands, the PAM dependence, and the guide-swap experiments that demonstrate programmability — is available at the source below.
Published online 28 June 2012