105of the prokaryote. As mentioned earlier, Cas1 and Cas2 play an important role in
spacer acquisition. They form a complex in which Cas2 forms one dimer, which
joins with two Cas1 dimers, in which the Cas1 depends on the Cas2 to bind the
CRISPR DNA [ 32 ]. Spacer selection, though, is not arbitrary; certain sequences are
present in the target that drive the spacer selection. The presence of a short
3–5-nucleotide long motif called protospacer adjacent motif (PAM) next to a potential
target is extremely important for target selection. PAM is very important to the pro-
karyote CRISPR as it helps in distinguishing between self and non-self [ 33 ]. In type
II-A, the signature protein, Cas9 nuclease, is tasked with the identification of PAM
in prospective targets [ 34 ].
6.3 History of CRISPR
The CRISPR locus or array was first isolated unintentionally by Ishino et al. [ 25 ]
from Escherichia coli while they were cloning the iap gene. The function or the
effect of such sequences were not known and understood at that time. For many
years, the existence of such direct repeats was just considered to be a quirk of the
bacterial genome. Multiple research groups then observed similar interrupted direct
repeats in the genome of multiple prokaryotes, giving rise to understanding that
CRISPR sequences occur in a lot of prokaryotic genomes. The real research into
understanding the use of CRISPR began in Danisco, a dairy company where
Philippe Horvath and Rodolphe Barrangou, while sequencing the Streptococcus
thermophilus genome, came across CRISPR repeats.
Initially, the reason behind the existence of such repeats was not understood, butas they sequenced multiple strains of the bacterium, it was theorized to be linked to
an immune system that would act as a defense mechanism against phage infection.
Since phage-based infection is a massive problem for the dairy industry as it needs
starter cultures to make cheese and yogurt, research into an immune system that
could stop phage infections was high on the agenda. As they confirmed that CRISPR
actually confers phage resistance, Barrangou et al. [ 23 ] later began to use the inher-
ent bacterial CRISPR system to immunize the strains against different phages. The
surviving bacteria were then used to create starter cultures that were immune to
phage attacks. This was very important as Danisco is part of DuPont and the parent
company owns a huge share of the global dairy market. Since they used improved
or modified bacterial strains to create cheese and yogurt, there is a good chance that
we have ingested CRISPR-based food. But, it is not to be considered a GMO since
no recombinant technology is involved.
But, scientists soon figured out that, although the CRISPR system works natu-rally in bacteria and archaea, the components of the system can be used to create
specific mutations in the genome of most organisms due to the precise targeting of
the CRISPR system. This led to massive interest in research on the CRISPR system
and its subsequent application in genome editing. The organism of choice was
Streptococcus pyogenes. Jinek et al. [ 8 ] characterized and engineered the type II
6 CRISPR: From Prokaryotic Immune Systems to Plant Genome Editing Tools