Clustered Regularly Interspaced Short Palindromic Repeats, known as CRISPR, are DNA segments within the genomes of bacteria and archaea. Within these arrays, short DNA sequences called spacers are found, typically ranging from 23 to 72 base pairs in length. These sequences serve as a genetic record of past encounters with invading genetic material, such as viruses or plasmids. This system allows these single-celled organisms to recall and respond to previously encountered threats.
Spacers in Microbial Defense
Spacers play a direct role in the natural immune system of bacteria and archaea, known as the CRISPR-Cas system. When a microbe encounters a foreign genetic element, like a virus, the spacer sequences within its CRISPR array are transcribed into short RNA molecules called CRISPR RNAs, or crRNAs.
These crRNAs combine with CRISPR-associated (Cas) proteins to form a surveillance complex. This complex actively patrols the cell, searching for foreign DNA sequences that precisely match the spacer sequence contained within the crRNA.
Upon locating a matching foreign DNA sequence, identified by an adjacent protospacer adjacent motif (PAM), the Cas proteins cleave the invading genetic material. This targeted destruction prevents the foreign DNA from replicating and harming the host cell. The precision of this recognition and cleavage mechanism allows the microbe to defend itself against specific invaders it has previously encountered.
How Microbes Acquire New Spacers
The acquisition of new spacers is a dynamic process that provides bacteria and archaea with adaptive immunity against new threats. When a microbe is infected by a novel virus or encounters other foreign genetic elements, specific Cas proteins are activated.
Cas1 and Cas2 proteins capture small fragments of invading DNA, often called “prespacers.” These fragments are then inserted into the microbe’s CRISPR array, becoming new spacers.
This integration process ensures that the newly acquired spacer sequence is flanked by existing repeat sequences within the CRISPR array. By adding these new genetic memories, the microbe updates its defense system, allowing it to recognize and neutralize the specific threat if it invades again. This mechanism highlights the adaptive nature of the CRISPR-Cas system, enabling microbes to evolve their immunity.
Spacers as Guides in Genetic Engineering
The natural function of CRISPR spacers as guides for Cas proteins has been harnessed to develop genetic engineering tools. Scientists have engineered synthetic guide RNAs (sgRNAs) that mimic the natural crRNA-tracrRNA complex found in CRISPR-Cas9 systems.
These synthetic sgRNAs contain a user-defined spacer sequence, which can be custom-designed to match almost any specific DNA sequence in a genome. The sgRNA then forms a complex with the Cas9 enzyme, directing it to the exact location in the target genome.
Once the sgRNA-Cas9 complex binds to the target DNA, guided by the complementary spacer sequence and a protospacer adjacent motif (PAM), the Cas9 enzyme introduces a double-strand break at that site. This targeted cut allows scientists to disrupt a gene, insert new genetic material, or make precise edits to the DNA sequence, offering control over genetic modification.