How does the CRISPR system carry out the exquisite process of catching invading phages and sending them to annihilation? The essential steps are summarized below.
Step 1: During the initial phage invasion of bacteria, phage DNA is shuffled to the bacterial CRISPR locus. Of note, CRISPR only cuts off a unique stretch of phage DNA to be stored in bacterial CRISPR “bank” called CRISPR arrays. Each “bank” deposit represents a unique phage sequence that bacteria can rely on to destroy the same phage in the future.
Which unique phage DNA does CRISPR select? The answer is “NGG”. “G” represents guanine, one of the four nucleotides that serve as the bases of nucleic acid (both DNA and RNA). “GG” is a guanine doublet. “N” designates any of the four nucleotides of A, T, G, or C. So, “NGG” could be “AGG”, “TGG”, “GGG”, or “CGG”.
After identifying the “NGG” site, CRISPR Cas9 excises phage DNA that starts with “NGG” and stores it in the CRISPR array. Cas9 is dubbed as the CRISPR scissors. CRISPR/Cas9 is also responsible for locating and eliminating future phages whose “NGG”-marked DNA sequence is stored in CRISPR arrays.
Step 2: During the subsequent phage invasion, the stored phage DNA in the CRISPR locus undergoes transcription into RNA, called CRISPR RNA, abbreviated as “crRNA”. Recall that DNA building blocks are A, T, G, and C, whereas RNA consists of A, U, G, and C, where “T” is substituted by “U”.
Step 3: crRNA then binds to a large protein complex, an example of which is known as “Cas9,” an acronym for “CRISPR-Associated 9,” a member of the Cas family.
