How and, lastly, the demonstration that the CRISPR–Cas9 system

How to construct CRISPR

The type II CRISPR system uses an endonuclease, Cas9,
which is guided by a single guide RNA (sgRNA) that specifically hybridizes and
induces a double-stranded break (DSB) at complementary genomic sequences 13. The discovery
of a short DNA sequence adjacent to the RNA-binding site, later termed the
protospacer-adjacent motif (PAM), as the CRISPR Cas mechanism for
discriminating self from non-self 17; the discovery of a small  transactivating CRISPR RNA (tracrRNA), which
directs the post-transcriptional processing and maturation of the CRISPR RNA
(crRNA) through sequence complementarity18; and, lastly, the demonstration that
the CRISPR–Cas9 system from S. thermophilus could function in Escherichia coli
and provide resistance against foreign plasmids14. On the basis of these findings about CRISPR–Cas9
biology, it was demonstrated that the Streptococcus pyogenes Cas9 protein can
bind to a tracrRNA–crRNA complex or to a designed, chimeric sgRNA to generate a
double-strand break (DSB) at a specific site of the target DNA in vitro 13, 15. Another report similarly showed that S. thermophilus
Cas9 could interact with the tracrRNA–crRNA complex to cut DNA 13. Demonstrations of the use of Cas9 and RNAs for
genome editing in vivo rapidly followed this seminal observation 16.

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The bacterial
genome contains regions with short repetitive stretches of DNA that are
separated by spacers. Researchers made the startling discovery that the spacers
are often composed of bits of foreign DNA and it transpired that bacteria use
it as a molecular memory of prior infection. When the same pathogen is
encountered again, the stretches of repeats and spacers are transcribed to form
CRISPR RNAs (crRNA). Together with a transactivating RNA (tracrRNA), it forms a
kind of GPS system for a series of CRISPR-associated (Cas) proteins that
function like molecular scissors, destroying the target DNA sequence in the
invader’s genome.



Transcription repression by CRISPRi

Bacteria lack the machinery for RNAi, and simple
platforms for targeted gene regulation in bacteria have been limited. The
utility of dCas9 for sequence-specific gene repression was first demonstrated
in E. coli as a technology called CRISPR interference (CRISPRi) 5. By pairing dCas9 with a sequence-specific
sgRNA, the dCas9–sgRNA complex can interfere with transcription elongation by
blocking RNA polymerase (Pol). It can also impede transcription initiation by
disrupting transcription factor binding 17, 18. Efficient
dCas9-mediated transcription repression in bacteria demonstrated the
possibility of using RNA-guided mechanisms for transcription repression and
activation in diverse organisms 19. CRISPR–Cas is currently divided into two major classes and
five types, of which type II is the most widely used for genome-engineering
applications 17.