Figure 1.

CRISPR/Cas9 mediated adaptive immunity in bacteria. A typical CRISPR locus consists of a leader sequence followed by an array of short identical repeats interspaced by short unique spacer sequences, as well as a set of CRISPR-associated (cas) genes (5). Preceding the cas genes is the tracrRNA, which encodes a non-coding RNA that is complementary to the repeats. During the acquisition stage, foreign DNA was cleaved into short DNA fragments (protospacers) and incorporated into the CRISPR array in chronological order of invasion as a spacer (6). Once integrated, the new spacer is transcribed with all other spacers into a pre-crRNA. The tracrRNA is transcribed separately and combines with the repeat sequence of the pre-crRNA to form a heterodimer. Then, the heterodimer RNA is cut by RNase III to form mature crRNA (7). When the same foreign DNA invades again, the mature crRNA-tracrRNA structure engages the Cas9 protein to form an RNP. RNP guides the Cas9 protein to recognize the PAM sequence (NGG for SpCas9) of the foreign DNA by matching the crRNA with the exogenous genes and performing site-specific double-strand cleavage at the three bases upstream, then the foreign DNA sequence is destroyed (6).