CRISPR
CRISPR-Cas9 is a programmable DNA-cutting system that researchers have adapted from bacterial immunity. It is the current gold standard for gene editing because it is simple, cheap, and very precise. The 2020 Nobel Prize in Chemistry recognized the development of CRISPR as a gene-editing tool.
The Bacterial Origin
In bacteria, CRISPR (clustered regularly interspaced short palindromic repeats) is an immune system against viruses. Bacteria store short snippets of past viral DNA in a special genomic region. When the virus attacks again, the bacterium transcribes these snippets into guide RNAs that direct a Cas nuclease to cut matching viral DNA.
Molecular biologists hijacked this system. By designing a guide RNA that targets any sequence of interest, Cas9 can be directed to cut any gene in any organism.
The Two Components
- Cas9: an RNA-guided endonuclease that cuts both DNA strands.
- Guide RNA (gRNA): a ~100-nucleotide RNA with a 20-nucleotide spacer that base-pairs with the target DNA sequence. The rest of the gRNA binds Cas9.
Target DNA must have a PAM sequence (typically NGG for the common SpCas9) immediately adjacent. The PAM is read by Cas9 and is not part of the gRNA pairing; it prevents Cas9 from cutting bacterial CRISPR arrays (which lack PAMs).
Editing Outcomes
Cas9 makes a blunt double-strand break (DSB) ~3 bp upstream of the PAM. The cell must repair this break. The repair pathway determines the editing outcome:
- Non-homologous end joining (NHEJ): the default pathway. The two broken ends are glued back together, often with small insertions or deletions (indels). A frame-shift indel in a coding region typically knocks out the gene. This is how CRISPR produces gene knockouts.
- Homology-directed repair (HDR): if a donor DNA template with homologous ends is provided, the cell can use it to repair the break precisely. This enables knock-in of specific edits, corrections of disease mutations, or insertion of tagged sequences.
Applications
- Research: creating knockout cell lines and animals. CRISPR has replaced older gene targeting methods almost entirely.
- Agriculture: drought-resistant crops, disease-resistant livestock.
- Therapeutics: the first FDA-approved CRISPR therapy, Casgevy (2023), treats sickle cell disease by editing hematopoietic stem cells. Several cancer and genetic disease trials are ongoing.
- Diagnostics: CRISPR-based detection systems (SHERLOCK, DETECTR) use Cas13 or Cas12 to detect specific nucleic acid sequences from pathogens.