Evolution of CRISPR in Prokaryotic Domain


As outlined previously, the CRISPR/Cas system was originally discovered by Nakata and his research group from Osaka University, Japan, in 1987 in prokaryotes. The researchers observed the clustered regularly interspaced short palindromic repeat (CRISPR) sequence adjacent to the iap gene in Escherichia coli K12 gram-negative bacteria. However, the function of CRISPR largely remained unknown. Later, the CRISPR sequences were also observed in other gram-negative and gram-positive bacteria, and in about 90% of the archaeal genome.

To date, the CRISPR/Cas system has not been reported in eukaryotic species. Different types of CRISPR/Cas systems have been reported in prokaryotes and archaea. They have been classified as Class I and Class 2. Type I and III CRISPR/Cas systems belong to the Class I category, while type II, IV, V, and VI CRISPR/Cas systems belong to Class 2. The well-research CRISPR/Cas9 discovered in Streptococcus pyogenes is a type II CRISPR system and belongs to the Class 2 category.

Although initially, the role of the CRISPR/Cas system in prokaryotes was poorly understood, subsequent research in the mid-2000s elucidated the role of the CRISPR sequences. In prokaryotes and archaea, CRISPR/Cas system functions as a component of their self-defence against attacking viruses and bacteriophages, thus conferring them adaptive immunity. 

The mechanism through which the CRISPR/Cas system functions depends upon its type and the class in which it belongs. However, in general, when bacteria is attacked by a phage, the CRISPR/Cas system gets activated to neutralise the threat in three key stages, namely, spacer acquisition, crRNA biogenesis, and target interception. CRISPR/Cas9 system has been studied extensively and the crystal structure of Cas9 endonuclease is well-documented. Thus, the role of CRISPR/Cas in prokaryotic self-defence will be explained using CRISPR/cas9 as an example.

Spacer acquisition: In this step, the Cas9 endonuclease along with Csn2 (a type IIA CRISPR-associated protein), and tracrRNA (trans-activating CRISPR RNA) of the bacteria cleaves off a distinct and characteristic sequence from the invading viral DNA. The target sequence (also known as the protospacer) and the site of cleavage are determined by the PAM that flanks the 3′ end of the viral DNA (target). Upon identifying the protospacer, the Cas9 binds to the target DNA and undergoes a conformational change to form an R-shaped loop. The target DNA is cleaved 3 bp upstream of its PAM region. The cleaved protospacer is integrated into the CRISPR array and incorporated into the bacterial genome.

crRNA biogenesis: Upon successful spacer acquisition, the CRISPR array is transcribed into 20- nucleotide (nt)-pre-CRISPR RNA (pre-crRNA). The tracrRNA being complementary in sequence to the pre-crRNA forms an RNA duplex with the pre-crRNA. Within the RNA duplex, the pre-crRNA matures to form crRNA with the help of RNase III. The tracrRNA-crRNA duplex generated is also known as the single guide RNA of 20-nt length. The single guide RNA (sgRNA) along with the Cas9 endonuclease forms the ribonucleoprotein complex that plays a crucial role in the final stage of target interception.

Target interception: In this stage, when the bacteria are again attacked by the same phage, the synthesised sgRNA guides the ribonucleoprotein complex to the target DNA with a complementary sequence. Target search and recognition are based on complementary base pairing between the crRNA spacer sequence (of the 20-nt sgRNA) and a protospacer in the target DNA in presence of the conserved PAM region adjacent to the target site. The PAM region in fact helps the CRISPR/cas9 system within the bacteria to distinguish between self and nonself sequences. Conserved PAM region is essential for target inception and a single mutation in the PAM region can help the virus to evade the bacterial self-defence mechanism. Once the complementary base pairing is achieved, the Cas9 endonuclease cleaves the target DNA, thereby neutralising the threat.

Although the CRISPR/Cas system was initially discovered in prokaryotes and archaea, advancements in research and technology have widened its scope of applications. CRISPR/Cas system is now extensively used as a cutting-edge genomic editing technology in the eukaryotic domain.