A protospacer is a specific segment of foreign genetic material, such as DNA from an invading virus or plasmid, that is recognized and targeted by a bacterial defense system. This system, known as CRISPR-Cas, identifies the protospacer as a threat, initiating a defense response.
The Role of Protospacers in the CRISPR-Cas System
Protospacers act as targets within the CRISPR-Cas bacterial immune system, enabling bacteria to defend against invading genetic elements. When a bacterium encounters foreign DNA, such as from a bacteriophage, the CRISPR-Cas system identifies these protospacers. This identification allows the bacterium to distinguish between its own genetic material and that of an invader.
The system relies on these protospacers to guide its defense machinery. By recognizing these specific sequences on the invading DNA, the CRISPR-Cas system mounts a targeted response. This ensures the bacterium only attacks foreign DNA, preventing damage to its own genome.
How Protospacers Are Identified
The CRISPR-Cas system locates protospacers using a molecular tag called the protospacer adjacent motif (PAM). This short DNA sequence, typically 2-6 base pairs in length, is located immediately next to the protospacer on the invading DNA. The Cas enzyme, such as Cas9, requires the presence of a PAM sequence to bind and cleave the target DNA.
For example, the Cas9 nuclease from Streptococcus pyogenes recognizes a 5′-NGG-3′ PAM sequence, where ‘N’ can be any nucleotide base. The Cas enzyme first identifies the PAM sequence, then partially unwinds the adjacent DNA to check for a match with its guide RNA. This guide RNA, derived from the bacterial CRISPR array, precisely base-pairs with the protospacer, ensuring accurate targeting.
The PAM sequence acts as a “self” versus “non-self” identifier, preventing the CRISPR-Cas system from attacking the bacterium’s own DNA. Bacterial CRISPR arrays do not contain these PAM sequences next to their integrated spacers. This two-step recognition—PAM binding followed by protospacer-guide RNA pairing—ensures the specificity of the defense mechanism.
The Journey from Protospacer to Spacer
When a bacterium survives an attack by a foreign genetic element, a fragment of the invading DNA, including the protospacer, is “captured” and integrated into the bacterium’s own CRISPR array. This process is called adaptation or acquisition. The Cas1 and Cas2 nucleases work together to identify and cut out a segment of the invading genetic material.
This captured protospacer is then inserted into the bacterial CRISPR array, becoming a “spacer” sequence. The new spacer serves as a genetic memory of the previous encounter. This allows the bacterium to “remember” specific invaders, enabling a faster and more effective defense upon subsequent infections.
The integration of the protospacer into the CRISPR array ensures that if the same invader reappears, the bacterial cell can quickly produce a complementary RNA sequence from the spacer. This RNA then guides the Cas nuclease to the invading DNA, leading to its destruction and protecting the bacterium.
Applying Protospacer Knowledge in Gene Editing
Understanding protospacers and their recognition by the CRISPR-Cas system has transformed gene editing, particularly with CRISPR-Cas9 technology. Scientists can leverage the natural protospacer-PAM recognition mechanism to program guide RNAs. These synthetic guide RNAs are designed to be complementary to specific DNA sequences in a target genome.
By designing a guide RNA that matches a desired genomic sequence and is located next to a suitable PAM, researchers can direct the Cas9 enzyme to a precise location. The Cas9 then creates a double-stranded break in the DNA at that site. This targeted DNA cleavage allows for specific modifications, including gene knockout, insertion, or correction of mutations.
This ability to precisely target and edit DNA sequences has opened numerous possibilities in research, disease modeling, and potential therapeutic applications. Repurposing this natural bacterial defense mechanism provides a tool for modifying genetic material in a wide range of organisms.