CRISPR-Cas9 System and Gene Editing

This assignment delves into the world of the CRISPR-Cas9 system, a powerful tool for genome editing. It discusses its mechanism, highlighting how it can be used to correct genetic mutations. Recent research is also explored, including studies that have successfully utilized CRISPR-Cas9 in various organisms and cells. The importance of this technology in advancing our understanding of genetics and its potential applications are also touched upon.

Running head: CRISPR Cas9
CRISPR cas9 in Genome Editing
Name of the Student
Name of the University
Author Note

1CRISPR Cas9
Introduction
Gene editing or genome editing is the technology that allows modification in the
DNA code of an organism. The alteration can be done by the addition, removal or alteration
of the genetic materials at specific locations of the genome. A recent method known as the
CRISPR Cas9 (abbreviation for clustered regularly interspaced short palindrome repeats-
CRISPR and CRISPR-associated protein 9-Cas9). This technology was adapted from the
bacterial system of gene editing that allows them to ‘remember’ viral genome by creating
arrays of genetic codes called the CRISPR arrays. Once the virus whose genetic code has
been added to the CRISPR array of the bacterial DNA enters the bacterial cell, cas9 enzymes
are then released to cut the DNA to disable the virus. This system is used by researches to
create short RNA strands that has a guide sequence, and binds to a particular DNA code in
the genome as well as cas9 enzyme. The RNA sequence then recognizes the specific DNA
sequence while the cas9 enzyme cuts the region. Enzymes like cpf1 can also be used instead
of Cas9 for cutting the DNA fragments. Once the specific sequence is cut from the genome, it
can be repaired with the correct code through the DNA repair process or replaced with a
customized DNA code (ghr.nlm.nih.gov 2018; Mali et al. 2013). This can be useful to detect
and correct aberrations in the gene that is often characteristics of diseases like cancer (Isola et
al. 1995). This technology is currently getting a lot of interest in the research on treatment for
human diseases like cystic fibrosis, hemophilia, sickle cell anemia, cancer and HIV (Schwank
et al. 2013; Park et al. 2015; Hsu et al. 2014).
Science and Methods Used 100 words
(Discussion about the methods of the study. Note: this will be a review of a secondary
research)

2CRISPR Cas9
The study by Hsu et al. (2014) analyzed the use of CRISPR cas9 genome editing
technology through secondary analysis of literature. Their studies discuss how CRISPR
technology can be used to design programmable nucleases, allow precise editing of the
genome, the structural architecture of Cas9, how this technique can be used in an eukaryotic
cell, how their recognition fidelity can be improved, and how the technology can be used in
research, medicine and biotechnology. The secondary research allows analysis of various
studies done on the topic to bring together a comprehensive understanding (Kothari 2004).
Data
Efficient and precise editing of genome using programmable nucleases:
Studies have shown that targeted double stranded breaks in the DNA can help genome
editing through homologous recombination (HR). Specific locus specific homologous
recombination (HR) has also been demonstrated using designer nucleases made from zinc
finger proteins (ZFP). Additionally, due to a lack of a template for repair for exogenous
homology, double stranded breaks can introduce deletions or insertions through non-
homologous pathways of joining of the ends (Hsu et al. 2014). Modifiable DNA binding
proteins like: zinc finger nuclease, transcription activator-like effectors (TALEs) and cas9
have been identified that can identify specific DNA sequences (Cong et al. 2013; Mali et al.
2013). The guide sequence in CRISPR array corresponds to the genomic sequence of the
phage, thereby providing antiviral resistance. Replacement of this sequence can then be made
with a sequence of interest, and thereby targeted by the cas9 nuclease. This customizable
DNA binding domain can allow modulation, as well as recruit desired changes like
transcriptional activation of a specific genetic locus (Mendenhall et al. 2013).
CRISPR cas 9 and Genome Editing:

3CRISPR Cas9
Studies by Sapranauskas et al. (2011) showed that type II CRISPR system can be
transferred, and transplanting the CRISPR locus from Streptococcus thermophilus to
Eschericia coli allowed the reconstitution of the CRISPR interface in the recipient cell.
Studies by Gasiunas et al. (2012) also showed that purified cas9 guided by guide RNA can be
used in vitro to target DNA sequences. Furthermore, RNA guide sequence is made by
combining trans-activating crRNA with target guide sequence and tracrRNA (Jinek et al.
2012). This engineered type II CRISPR systems can allow genome editing of mammalian
cells, and multiple guide RNA to target multiple genetic codes simultaneously(Cong et al.
2013; Mali et al. 2013; Sander et al. 2014).
Cas9 structure and domain:
Electromagnetic reconstructions of cas9 enzyme of Streptococcus pyogenes shows a
significant reshuffling with apo-cas9 (not bound to nucleic acid) and cas9 (in a conjunction
with crRNA and tracr RNA) that forms a core path for RNA-DNA hybrid formation (Jinek et
al. 2014). Research by Nishimasu et al. (2014) pointed that the cas9 domain consisted of
alpha helical recognition (REC) lobe as well as a nuclease lobe. This suggests Spcas9
unbound to guide RNA or target DNA has autoinhibited conformation and the active site of
the enzyme is blocked. The guide RNA acts as scaffold surrounding which the folding of
cas9 can occur to organize the domains (Nishimasu et al. 2014).
Diversity of cas9
Case 9 is related exclusively to the type II CRISPR locus and functions as a typical
type II gene. The type II locus is further categorized into 3 subtypes: IIa, IIb and IIc
(Chylinski et al. 2013). This loci generally consists of cas9, cas1, cas2 gene apart from the
CRISPR array and tracrRNA, however, IIC locus contains minimal cas genes, and IIA and
IIB contains additional genes like csn2 and cas4 (Chylinski et al. 2013). However, even with

4CRISPR Cas9
the evident diversity of genetic assembly, cas9 have structurally similar domains (Fonfara et
al. 2013). The type II CRISPR have been identified only in bacterial systems, not like in the
case of type I and III, which are found in bacteria and archaea (Chylinski et al. 2013).
Cas9 target range and search mechanism
Molecule imaging studies shows that the complex of cas9-crRNA-tracrRNA
associates with protospacer-adjacent motif first (PAM) at the 3’ end of the target DNA
thereby dictating the search method of cas9, and discriminate self versus non self, and
facilitate the formation of cleavage conformation of the enzyme (Shah et al. 2013). This
binding triggers the nuclease activity of the enzyme, and is evidenced by its domain
flexibility (Sternberg et al. 2014; Nishimasu et al. 2014). The PAM has a high specificity
within each ortholog even among same species. The range of targets of the cas9 toolkit
however can be expanded by the inclusion of additional PAM and allow orthogonal genome
editing (Chylinski et al. 2013; Fonfara et al. 2013). However, the specific need of PAM is
modified by replacing the PAM interacting domain from one species to another as shown by
Nishimasu et al. 2014.
CRISPR cas9 in Eukaryotic cell
Cas9 derived from Streptococcus pyogenes (SpCas9) was used successfully
for editing the genome of various types of cells like bacteria, pig human cell lines,
mouse, roundworm, zebra fish, yeast, fruit fly, rat, monkeys, and even common
crops (Sander and Joung 2014). Studies by Niu et al. (2014) showed the use of
SpCas9 to induce multiplex mutations in monkeys. CrisprCas9 can also act in
parallel to target and cleave multiple sequences (Garneau et al. 2010). This ability can

5CRISPR Cas9
be harnessed to indi8ce multiple perturbations, allowing multiplex editing in mammalian
cells (Hsu et al. 2014).
Evaluation of Data
Rapid Generation of Cellular and Animal Models
Genome editing by cas9 have allowed accelerated the generation of transgenetic
models, expanding the scope of biological studies beyond traditional animal models. The
technology also helps to understand the causative functions of variations in genes in the
disease aetiology, through the reiteration the mutations in genetic pool (Sander and Joung
2014). Novel transgenic animal models can also be developed with mutations introduced at
specific locus or corrected by in-vivo or ex vivo gene correction techniques (Niu et al. 2014;
Schwank et al. 2013). Considering that genetic aberrations can lead to cancers, such
technology can be useful for cancer research.
Functional Genomic Screens
The ability to change several target genomes parallel to each other, a genome wide
functional screen can be developed that can help to find genetic codes that underlie specific
charactersistsics of interest (Hsu et al. 2014). Studies by Wang et al. (2014) and Shalem et al.
(2014) showed thousands of genetic elements can be altered parallel, by the insertion of
sgRNA directed towards all genes, along with cas9 or in a cell that already produces cas9
enzyme in human cell lines. This can help to create perturbations in non coding genetic
elements as well as dissect the function of complex genetic elements by tiled micro deletions
of genes. It can therefore be used to map large uncharacterized regions of chromosomes (Hsu
et al. 2014). This can be another useful technique to identify genes that can lead to diseases.
Transcriptional Modulation

6CRISPR Cas9
The machinery of RNA polymerase can be modulated sterically through the binding
of dCas9 molecules to the DNA elements. This property can be used to convert cas9 into a
transcriptional activator (Qi et al. 2013). A decent transcriptional upregulation may be
attained by targeting cas9 activators in each sgRNA for a promoter gene (Konerman et a.
2013). Such aspects can be used to understand the modulation of the transcription machinery,
and study how transcription factors control the expression of genes (Hsu et al. 2014).
Epigenetic Control
Highly complex epigenetic states create a highly dynamic landscape of complex
genomic functions. Epigenetic machinery that alters histones acts as transcriptional regulators
and play vital role in biological function. Specific genomic loci introduced through different
enzymes were able to induce methylation of the DNA or acetylation of the histones (Hsu et
al. 2014). This can be a useful mechanism to study the epigenetic factors associated with
diseases. This can also be used to understand the epigenetic modifications that shape the
regulatory networks of genomes. However, careful characterization of the off target functions
and crosstalk between the different genomic domains shill needs to be characterized in detail
(Hsu et al. 2014).
Live Imaging
Studies show that the function of the genes is controlled by the spatial positioning of
the structural as well as functional components in a cell, and this system can be either
suppressed or amplified dynamically. Genomic loci that are far apart can be brought close to
each other thereby allowing long range trans interactions. This allows a robust way of
visualizing the DNA of live cells by analysing the interplay between genes through dynamic
states of the chromatin. Labeling of DNA with cas9 enable capturing of live processes and is
a good alternative to DNA FSH (Chen et al. 2013). This can be used to understand the

7CRISPR Cas9
processes of diseased as well as normal cells to understand the differences in the cell
physiologies and metabolic pathways (Hsu et al. 2014_.
Inducible Regulation of Cas9
Study of the structure of cas9 molecule, can help to to split it into two component
subunits and then aid their reassembly through light inducible heterodimeric domains, this
can allow systemic control of cas 9 in patients as well as animal models (Hsu et al. 2014).
This can allow precise tuning of the cas9 molecule to induce specific changes in the genome
of the test cell.
Conclusion
CRISPR cas 9 in a novel technology that allows genomic editing with high fidelity
and specificity. This technology was derived from the bacterial system that provided them
protection against viral pathogens. The system works by incorporating the viral DNA into the
bacterial genomic locus called the CRISPR array. The CRISPR array also codes for specific
enzymes like cas9 that recognizes specific regions of the DNA, based on the guide RNA
codes. Genomic editing is done by synthesizing a guide RNA code complementary of DNA
target sequence, and joining the cas9 molecule to the guide sequence. Once the RNA
sequence hybridizes with the DNA target, the cas9 enzyme cuts the DNA segment. Later, the
missing fragment can be replaced by any genomic locus of choice. This technology has been
studied extensively for the treatment of several genetic diseases and has shown promising
results in animal test and human cell lines. The ability of CRISPR cas9 to induce multiple
genetic changes simultaneously, modulate transcription mechanisms and analyze epigenetic
modifications of genes, has made it possible to study both simple and complex genomic
functions. This has a vital implication in the understanding of how different disease changes

8CRISPR Cas9
the genetic as well as epigenetic functions. The technology also provides a breakthrough way
of visualizing live cells, and can be used in the study of cellular functions in greater detail.

9CRISPR Cas9
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10CRISPR Cas9
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11CRISPR Cas9
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12CRISPR Cas9
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