136
possessing Protospacer Adjacent Motif (PAM) (Brouns et al. 2008; Waters and
Storz 2009; Deveau et al. 2010; Deltcheva et al. 2011; Bolotin et al. 2005).
• PAM is short length double stranded nucleotide chain usually 2–5 base pair.
Different PAM sequences are found in different bacteria. These sequences help
the Cas9 machinery to locate the phage DNA and aid in distinguishing between
host DNA and phage DNA (Mojica et al. 2009; Strich Chertow 2019; Shah et al.
2013; Tyson and Banfield 2008; Horvath et al. 2008; Deveau et al. 2008;
Andersson and Banfield 2008; Pride et al. 2011; Yosef et al. 2012; Díez-Villaseñor
et al. 2013; Swarts et al. 2012; Goren et al. 2012; Datsenko et al. 2012).
• To reach the site of action, Cas9 machinery requires the help of guide RNA. These
sequences escort Cas9 to the target DNA (for genome editing). DNA cleavage
takes place only when the target is followed by PAM sequence. In case of any
mutation in PAM or incompatibility between spacer and foreign DNA, the DNA
cleavage will not take place and host is prone to infection. The specificity of Cas
proteins machinery is important in order to develop an active adaptive immune
response by bacteria against bacteriophage (Tsai et al. 2015; Shah et al. 2009;
Shah et al. 2013; Pourcel et al. 2005; Tyson and Banfield 2008; Horvath et al.
2008; Deveau et al. 2008; Andersson and Banfield 2008; Pride et al. 2011; Yosef
et al. 2012; Díez-Villaseñor et al. 2013; Swarts et al. 2012; Goren et al. 2012;
Datsenko et al. 2012; Mojica et al. 2009; Lillestøl et al. 2009; Shah et al. 2013;
Anders et al. 2014; Esvelt et al. 2013; Zhang et al. 2014a).
6.4 Management of Antimicrobial Resistance Using
CRISPR-Cas System
Antimicrobial resistance is a condition when the microbes (such as bacteria, fungi,
viruses and parasite) develop resistance/ immunity against the antimicrobial agents.
One of the causes of antimicrobial resistance is mutations. The sequences causing
antimicrobial resistance in bacteria can be edited in order to re-sensitize the bacteria
towards antibiotics. There are many nucleases which are used for gene editing like
homing endonuclease (meganuclease), transcription activator like effector (TAE)
and Zinc finger nucleases (ZNF). However, there are certain limitations associated
with these techniques which are listed in Table 6.2.
CRISPR-Cas9 machinery can be an interesting and promising gene editing tool
to overcome antimicrobial resistance, as shown in Fig. 6.2. CRISPR-Cas9 is simple
and unique amongst the CRISPR systems as only one protein is sufficient to cause
gene silencing (Shabbir et al. 2019; Kim et al. 2015; Fernandes et al. 2019). As a
result, CRISPR-Cas9 has been the tool of choice for gene editing. This potential
was first reported by Doudna and Charpentier research groups (Jinek et al. 2012). A
team of researchers led by Zhang were amongst the first to use CRISPR Cas9 system for gene editing in eukaryotes cells (Cong et al. 2013). In CRISPR type II
mechanism, guide DNA facilitates the Cas9 machinery to identify the target
A. P. Sarma et al.
possessing Protospacer Adjacent Motif (PAM) (Brouns et al. 2008; Waters and
Storz 2009; Deveau et al. 2010; Deltcheva et al. 2011; Bolotin et al. 2005).
• PAM is short length double stranded nucleotide chain usually 2–5 base pair.
Different PAM sequences are found in different bacteria. These sequences help
the Cas9 machinery to locate the phage DNA and aid in distinguishing between
host DNA and phage DNA (Mojica et al. 2009; Strich Chertow 2019; Shah et al.
2013; Tyson and Banfield 2008; Horvath et al. 2008; Deveau et al. 2008;
Andersson and Banfield 2008; Pride et al. 2011; Yosef et al. 2012; Díez-Villaseñor
et al. 2013; Swarts et al. 2012; Goren et al. 2012; Datsenko et al. 2012).
• To reach the site of action, Cas9 machinery requires the help of guide RNA. These
sequences escort Cas9 to the target DNA (for genome editing). DNA cleavage
takes place only when the target is followed by PAM sequence. In case of any
mutation in PAM or incompatibility between spacer and foreign DNA, the DNA
cleavage will not take place and host is prone to infection. The specificity of Cas
proteins machinery is important in order to develop an active adaptive immune
response by bacteria against bacteriophage (Tsai et al. 2015; Shah et al. 2009;
Shah et al. 2013; Pourcel et al. 2005; Tyson and Banfield 2008; Horvath et al.
2008; Deveau et al. 2008; Andersson and Banfield 2008; Pride et al. 2011; Yosef
et al. 2012; Díez-Villaseñor et al. 2013; Swarts et al. 2012; Goren et al. 2012;
Datsenko et al. 2012; Mojica et al. 2009; Lillestøl et al. 2009; Shah et al. 2013;
Anders et al. 2014; Esvelt et al. 2013; Zhang et al. 2014a).
6.4 Management of Antimicrobial Resistance Using
CRISPR-Cas System
Antimicrobial resistance is a condition when the microbes (such as bacteria, fungi,
viruses and parasite) develop resistance/ immunity against the antimicrobial agents.
One of the causes of antimicrobial resistance is mutations. The sequences causing
antimicrobial resistance in bacteria can be edited in order to re-sensitize the bacteria
towards antibiotics. There are many nucleases which are used for gene editing like
homing endonuclease (meganuclease), transcription activator like effector (TAE)
and Zinc finger nucleases (ZNF). However, there are certain limitations associated
with these techniques which are listed in Table 6.2.
CRISPR-Cas9 machinery can be an interesting and promising gene editing tool
to overcome antimicrobial resistance, as shown in Fig. 6.2. CRISPR-Cas9 is simple
and unique amongst the CRISPR systems as only one protein is sufficient to cause
gene silencing (Shabbir et al. 2019; Kim et al. 2015; Fernandes et al. 2019). As a
result, CRISPR-Cas9 has been the tool of choice for gene editing. This potential
was first reported by Doudna and Charpentier research groups (Jinek et al. 2012). A
team of researchers led by Zhang were amongst the first to use CRISPR Cas9 system for gene editing in eukaryotes cells (Cong et al. 2013). In CRISPR type II
mechanism, guide DNA facilitates the Cas9 machinery to identify the target
A. P. Sarma et al.