CRISPR
CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats was
discovered about 30 years ago when haloarchaea Haloferax mediterranei DNA was being
researched (Mojica and Rodriguez-Valera, 2016). Repeated genetic sequences that could
be read the same way forwards and backwards (a palindrome) with different spacers
between these segments that did not have any relation to any known protein were
noticed on sequencing films. A key breakthrough in 2007 found that these palindromic
repeats form part of the immune system of most prokaryotes (Barrangou et al., 2007).
This study showed that when prokaryotes were infected by bacteriophages, the short
repeats were separated with new spacers derived from the genome of the attacking
bacteriophage. Removing these spacers from the virus also destroys the virus in the
process. As part of the prokaryotic genome, these spacer sequences can be transcribed
into a guide RNA that has the same sequence as parts of the viral genome and then base-
pairs with these genes if they come into contact again, giving the prokaryote a genetic
memory of the phage infection. However, it was not until 2012 that the concept of their
gene editing function was fully understood by Jennifer Doudna and Emmanuelle
Charpentier (Reece et al., 2018). This essay will provide an overview of the mechanisms
behind this system as well as a brief look at its potential to be used as a gene engineering
tool, the future prospects and challenges facing it within the field of microbiology.
There are three main CRISPR systems, distinguishable by their different nucleases called Cas
(CRISPR-associated) (Makarova and Koonin, 2015). The Cas9 protein forms part of the type
two system, the Cas3 in the type one system and the Cas10 in the type ten system. All three
systems contain the major components of the prokaryotic immune system. In this essay I
will be focusing on the type two system involving the Cas9 protein. The CRISPR-Cas9 system
consists of a complex containing the Cas9 nuclease and guide RNA (consisting of two distinct
segments of DNA- CRISPR RNA - crRNA and transactivating CRISPR RNA – tracrRNA, which
form a duplex molecule) (Campbell et al., 2017) (see figure 1). This complex can enter a cell
and the guide RNA guides the Cas9 protein to a particular genetic sequence. Cas9 has active
sites which can cleave sequences base-paired with the guide RNA, resulting in double
, stranded breaks which the cell can repair in two different ways. In the case of infection by
bacteriophage DNA, the foreign DNA will be cleaved and destroyed.
The two main types of repair are non-homologous end joining (NHEJ) and homology-
directed repair (HDR) (Ahern, Rajagopal and Tan, 2012). NHEJ simply ligates the DNA back
together but it is prone to mistakes causing insertions and deletions of nucleotides. This
could result in a frameshift mutation if the number of nucleotides inserted or deleted is not
divisible by three – terminating the function of the protein. HDR replaces the targeted genes
with another sequence, using a DNA donor template with regions of homology either side of
the desired sequence which is introduced to the cell along with the CRISPR components.
This incorporation of new material into a genome is known as a knock-in.
There are also a few creative variations on the CRISPR-Cas9 system. One of these is known
as CRISPR interference. This variation uses a mutated Cas9 endonuclease so that it pairs
with a target sequence but no longer cleaves any DNA (Qi et al., 2013). This binding blocks
access to the promoter so the transcription of the downstream gene is prevented. This
means that specific genes can be inactivated without altering the DNA sequence. Another
variation is called CRISPR activation. This variation also uses a disabled Cas9, but here it is
bound to a transcriptional activation domain (Ahern, Rajagopal and Tan, 2012). The guide
RNA places the Cas9 activation domain in a place where it can enhance a particular
promotor and activate target genes. Other variations include attachment of enzymes that
modify histones or DNA methylases to the inactive Cas9 or DNA methylases (Ahern,
Rajagopal and Tan, 2012). This causes the DNA histones to be modified or the DNA to be
methylated wherever the guide RNA positions the Cas9.
So, what are the current and future applications of CRISPR? CRISPR has transformed the
field of genome engineering as it is a simple and relatively cheap means of altering DNA in
prokaryotes as well as other organisms, including humans (Luo, Leenay and Beisel, 2016). It
holds potential to have the ability to cure debilitating genetic diseases such as muscular
dystrophy and sickle-cell anaemia, as seen in preclinical trials (Prabhune, 2018). Within the
field of microbiology there are many new and exciting ways in which this new technology
CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats was
discovered about 30 years ago when haloarchaea Haloferax mediterranei DNA was being
researched (Mojica and Rodriguez-Valera, 2016). Repeated genetic sequences that could
be read the same way forwards and backwards (a palindrome) with different spacers
between these segments that did not have any relation to any known protein were
noticed on sequencing films. A key breakthrough in 2007 found that these palindromic
repeats form part of the immune system of most prokaryotes (Barrangou et al., 2007).
This study showed that when prokaryotes were infected by bacteriophages, the short
repeats were separated with new spacers derived from the genome of the attacking
bacteriophage. Removing these spacers from the virus also destroys the virus in the
process. As part of the prokaryotic genome, these spacer sequences can be transcribed
into a guide RNA that has the same sequence as parts of the viral genome and then base-
pairs with these genes if they come into contact again, giving the prokaryote a genetic
memory of the phage infection. However, it was not until 2012 that the concept of their
gene editing function was fully understood by Jennifer Doudna and Emmanuelle
Charpentier (Reece et al., 2018). This essay will provide an overview of the mechanisms
behind this system as well as a brief look at its potential to be used as a gene engineering
tool, the future prospects and challenges facing it within the field of microbiology.
There are three main CRISPR systems, distinguishable by their different nucleases called Cas
(CRISPR-associated) (Makarova and Koonin, 2015). The Cas9 protein forms part of the type
two system, the Cas3 in the type one system and the Cas10 in the type ten system. All three
systems contain the major components of the prokaryotic immune system. In this essay I
will be focusing on the type two system involving the Cas9 protein. The CRISPR-Cas9 system
consists of a complex containing the Cas9 nuclease and guide RNA (consisting of two distinct
segments of DNA- CRISPR RNA - crRNA and transactivating CRISPR RNA – tracrRNA, which
form a duplex molecule) (Campbell et al., 2017) (see figure 1). This complex can enter a cell
and the guide RNA guides the Cas9 protein to a particular genetic sequence. Cas9 has active
sites which can cleave sequences base-paired with the guide RNA, resulting in double
, stranded breaks which the cell can repair in two different ways. In the case of infection by
bacteriophage DNA, the foreign DNA will be cleaved and destroyed.
The two main types of repair are non-homologous end joining (NHEJ) and homology-
directed repair (HDR) (Ahern, Rajagopal and Tan, 2012). NHEJ simply ligates the DNA back
together but it is prone to mistakes causing insertions and deletions of nucleotides. This
could result in a frameshift mutation if the number of nucleotides inserted or deleted is not
divisible by three – terminating the function of the protein. HDR replaces the targeted genes
with another sequence, using a DNA donor template with regions of homology either side of
the desired sequence which is introduced to the cell along with the CRISPR components.
This incorporation of new material into a genome is known as a knock-in.
There are also a few creative variations on the CRISPR-Cas9 system. One of these is known
as CRISPR interference. This variation uses a mutated Cas9 endonuclease so that it pairs
with a target sequence but no longer cleaves any DNA (Qi et al., 2013). This binding blocks
access to the promoter so the transcription of the downstream gene is prevented. This
means that specific genes can be inactivated without altering the DNA sequence. Another
variation is called CRISPR activation. This variation also uses a disabled Cas9, but here it is
bound to a transcriptional activation domain (Ahern, Rajagopal and Tan, 2012). The guide
RNA places the Cas9 activation domain in a place where it can enhance a particular
promotor and activate target genes. Other variations include attachment of enzymes that
modify histones or DNA methylases to the inactive Cas9 or DNA methylases (Ahern,
Rajagopal and Tan, 2012). This causes the DNA histones to be modified or the DNA to be
methylated wherever the guide RNA positions the Cas9.
So, what are the current and future applications of CRISPR? CRISPR has transformed the
field of genome engineering as it is a simple and relatively cheap means of altering DNA in
prokaryotes as well as other organisms, including humans (Luo, Leenay and Beisel, 2016). It
holds potential to have the ability to cure debilitating genetic diseases such as muscular
dystrophy and sickle-cell anaemia, as seen in preclinical trials (Prabhune, 2018). Within the
field of microbiology there are many new and exciting ways in which this new technology
