132
2005; Mojica et al. 2005; Bolotin et al. 2005). Later, first experimental evidence
claiming CRISPR as adaptive immunity was published (Hsuet al. 2014). It was
found that CRISPR locus comprised of DNA sequences obtained from the previous
invading viruses in Streptococcus thermophilus (Hsu et al. 2014; Pennisi 2013).
6.3 Mechanism of CRISPR-Cas Machinery
CRISPR mechanism of degrading foreign genome involves primarily three steps, as
shown in Fig. 6.1.
6.3.1 Integration of Spacer Sequencer in CRISPR Array after
Recognition (Spacer Acquisition)
• The primary step during the encounter of bacteria with virus is to arrest viral
DNA and integrate it in spacer array as a spacer. Presence of proteins like Cas1
and Cas2 in Cas network implies their involvement in integration of spacer in
CRISPR locus (Aliyari and Ding 2009; Dugar et al. 2013; Hatoum-Aslan et al.
2011; Yosef et al. 2012; Swarts et al. 2012).
• Two dimers of Cas1 protein linked by dimer of Cas2 protein forms a multiplex.
Staging of double strand DNA (dsDNA) is done by Cas2 protein and Cas1 protein helps in addition of spacer in the array (Nuñez et al. 2014). Recent spacers
are generally inserted at the starting of the CRISPR adjacent of leader sequence
facilitating the progressive track of viral infections. Integration host factor (IHF)
found in E. coli is a protein similar to histone which attaches with the leader
sequence and is responsible for the precise integration of spacer in the CRISPR
array (Nuñez et al. 2014).
6.3.2 Development of crRNA (Transcription Facilitated by
RNA Polymerase)
• Transcription CRISPR array: A typical CRISPR locus in a type II CRISPRCas system comprises a leader sequence, an array of repetitive sequences interspaced by spacer (segments of foreign DNA from previous invaders) as well as a
set of CRISPR-associated (cas) genes. CRISPR array undergoes transcription
and an unprocessed precursor CRISPR RNA (pre-crRNA) is obtained which is
further subjected to post transcriptional processing (Terns and Terns 2011;
Brouns et al. 2008; Hale et al. 2009).
• Base pairing of tract-RNA with pre-crRNA: Preceding the cas operon is the
trans-activating CRISPR RNA (tract-RNA) gene, which encodes a unique nonA. P. Sarma et al.
2005; Mojica et al. 2005; Bolotin et al. 2005). Later, first experimental evidence
claiming CRISPR as adaptive immunity was published (Hsuet al. 2014). It was
found that CRISPR locus comprised of DNA sequences obtained from the previous
invading viruses in Streptococcus thermophilus (Hsu et al. 2014; Pennisi 2013).
6.3 Mechanism of CRISPR-Cas Machinery
CRISPR mechanism of degrading foreign genome involves primarily three steps, as
shown in Fig. 6.1.
6.3.1 Integration of Spacer Sequencer in CRISPR Array after
Recognition (Spacer Acquisition)
• The primary step during the encounter of bacteria with virus is to arrest viral
DNA and integrate it in spacer array as a spacer. Presence of proteins like Cas1
and Cas2 in Cas network implies their involvement in integration of spacer in
CRISPR locus (Aliyari and Ding 2009; Dugar et al. 2013; Hatoum-Aslan et al.
2011; Yosef et al. 2012; Swarts et al. 2012).
• Two dimers of Cas1 protein linked by dimer of Cas2 protein forms a multiplex.
Staging of double strand DNA (dsDNA) is done by Cas2 protein and Cas1 protein helps in addition of spacer in the array (Nuñez et al. 2014). Recent spacers
are generally inserted at the starting of the CRISPR adjacent of leader sequence
facilitating the progressive track of viral infections. Integration host factor (IHF)
found in E. coli is a protein similar to histone which attaches with the leader
sequence and is responsible for the precise integration of spacer in the CRISPR
array (Nuñez et al. 2014).
6.3.2 Development of crRNA (Transcription Facilitated by
RNA Polymerase)
• Transcription CRISPR array: A typical CRISPR locus in a type II CRISPRCas system comprises a leader sequence, an array of repetitive sequences interspaced by spacer (segments of foreign DNA from previous invaders) as well as a
set of CRISPR-associated (cas) genes. CRISPR array undergoes transcription
and an unprocessed precursor CRISPR RNA (pre-crRNA) is obtained which is
further subjected to post transcriptional processing (Terns and Terns 2011;
Brouns et al. 2008; Hale et al. 2009).
• Base pairing of tract-RNA with pre-crRNA: Preceding the cas operon is the
trans-activating CRISPR RNA (tract-RNA) gene, which encodes a unique nonA. P. Sarma et al.