How do CRISPR-Cas systems recognize target DNA?

CRISPR-Cas systems recognize target DNA through a two-part mechanism that combines short protein-mediated sequence sensing with RNA-guided base pairing. Bacteria and archaea use CRISPR arrays as adaptive memory: short fragments of invading phage or plasmid DNA, called spacers, are stored in the host genome and transcribed as guide RNAs. Early identification of repetitive sequences and the adaptive role of CRISPR was reported by Francisco Mojica at University of Alicante, which set the stage for later mechanistic studies. Molecular work has since shown that recognition depends on a specific short motif in the target DNA and on precise hybridization between the guide RNA and the protospacer.

Protospacer adjacent motif and self–nonself discrimination
A critical element for target recognition is the protospacer adjacent motif, or PAM, a short DNA sequence immediately next to the complementary protospacer. The PAM is read directly by the Cas protein before RNA-DNA pairing proceeds, and it prevents self-targeting because the host CRISPR array lacks the appropriate PAM. For example, the widely used Streptococcus pyogenes Cas9 requires an NGG PAM, a specificity characterized in foundational work by Jennifer Doudna at University of California Berkeley and Emmanuelle Charpentier at Max Planck Unit for the Science of Pathogens. Different Cas proteins recognize different PAMs, shaping which sequences can be targeted across microbial diversity.

Guide RNA pairing and nuclease activation
Once a compatible PAM is engaged, the guide RNA forms an R-loop by base pairing with the complementary DNA strand. Successful pairing in a short PAM-proximal "seed" region stabilizes the complex, triggers conformational rearrangements in the Cas protein, and activates nuclease domains that cleave the DNA. In Cas9, distinct nuclease domains cut each DNA strand to produce a double-strand break. Other effectors such as Cas12 require different PAMs and undergo single-stranded cleavage activity after R-loop formation, while Cas13 targets RNA rather than DNA. Structural and biochemical studies from multiple groups including work translated to eukaryotic systems by Feng Zhang at Broad Institute have elucidated these transitions from PAM sensing to RNA-guided cleavage.

Causes, consequences, and broader relevance
The evolutionary cause for these recognition mechanisms is the arms race between microbes and their mobile genetic elements. Spacer acquisition and PAM-dependent interference provide adaptive, sequence-specific immunity. Consequences of this molecular logic extend beyond microbiology: the same rules that determine targeting specificity in nature govern off-target risk in genome editing applications, influence design of antiviral strategies in animals and plants, and raise environmental and ethical questions about gene drives and germline modification. Cultural and territorial considerations shape deployment and regulation, as research and applications advance at different paces in academic institutions, industry, and national regulatory systems. Understanding PAM requirements, seed-region sensitivity, and conformational checkpoints is therefore essential both for interpreting microbial ecology and for responsible use of CRISPR technologies in medicine, agriculture, and conservation.