CRISPR-Cas9: A Potential Tool for Genome Editing

1. Introduction

1.1. Origins

In the year 1987, a team of Japanese scientists were the first to describe an unusual locus

found in the E. coli genome, adjacent to the iap gene, having short palindromic repeats interspersed

by similarly sized non-repetitive DNA spacers (Fig. 1) [1]. This clustered, regularly interspersed,

short palindromic repeat locus is therefore termed “CRISPR”. Nearly a decade later, up to forty

percent of sequenced bacteria and ninety percent of archaea were found to harbor this CRISPR locus

[2] [3].

In the year 2002, bacterial strains that survived bacteriophage infection were observed to

express the CRISPR loci, suggesting that the particular region may have a role in prokaryotic adaptive

immunity [4]. This hypothesis was subsequently confirmed when phage-resistant bacterial strains had

specific CRISPR loci spacers disrupted, they acquired susceptibility to phage infection, while

insertion of novel spacers into wild type stains demonstrated acquired resistance [5].

The proteins involved in the prokaryotic immune system were found to be conserved and

encoded in close proximity to the CRISPR locus (Fig. 1) [6]. As such, these proteins are known as

CRISPR-associated, or in short, “Cas”.

1.2. Mechanism of Action

The bacterial CRISPR-based acquired immunity (Fig. 1) encompasses three processes,

namely, spacer addition, CRISPR-RNA (crRNA) maturation and target elimination [7][8][9].

Upon initial exposure to foreign phage DNA, bacterial Cas1 locates the DNA’s unique

protospacer adjacent motif (PAM) and cuts a short DNA fragment (protospacer) that is directly beside

[7]. Integration of this protospacer into the CIRSPR locus is directed by the Cas1-Cas2 complex [7].

Successful integration of protospacers into the host genome are thereafter referred to as spacers.

A pre-crRNA is a long mRNA transcript of the CRISPR locus containing an array of spacer

and repeat sequences. It hybridizes with multiple trans-activating crRNAs (tracrRNA) to form RNA

duplex structures that are targeted for cleavage by RNase III [10]. The cleaved, mature crRNA,

encodes for a particular spacer and repeat sequence which remains hybridized to a tracrRNA, and this

short duplex is called the guide RNA (gRNA) [7][8].

Each gRNA has a unique spacer sequence that recognizes its complementary protospacer of

the phage DNA [7][8]. When an immunized bacterial cell reencounters the same phage DNA, the

appropriate gRNA guides Cas9, an endonuclease, to specifically target and eliminate the invading

DNA by inducing a site-specific double strand break (DSB) [7][8]. The PAM ensures that Cas9

complexes locate the correct protospacer sequence [7]. As the PAM is only found on invading phage

DNA, the DSB mechanism is able to discriminate self from non-self [7].

Figure 1: Bacterial CRISPR-based acquired immunity. Phase 1 (immunization): Initial injection of phage double strand DNA into a

bacterial cell followed by Cas1-Cas2 complex (large teal oval with associated grey ovals) excision of the protospacer (yellow rectangle)

from the phage DNA. The Cas1-Cas2 complex inserts the protospacer into the Type II CRISPR locus region containing short palindromic

repeats (black) and novel spacers derived from other foreign phage DNA (purple, green and red rectangles). Phase 2 (immunity): Pre-crRNA

(multi-colored linear mRNA) and tracrRNA (purple stem-loop mRNA) are transcribed and processed into guide RNA (mature crRNA-

tracrRNA duplex) which subsequently guides Cas9 (small teal oval) to cleave the invading DNA. (Figure adapted from [9])

1.3. Tool for Genome Editing

There are four major tools capable of inducing site-specific double strand breaks (DSB); zinc

finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease and

CRISPR-Cas complex [11]. Any of these platforms may be programmed for DSB-induced genome

editing in prokaryotes and eukaryotes by exploiting their endogenous DNA repair mechanisms [11].

Breaks in the genome are remediated via one of two main repair mechanisms; non-

homologous end-joining (NHEJ) or homology-directed repair (HDR) [11]. The NHEJ pathway does

not utilize a template to flank the break region, hence, there is random insertion and/or deletions

(indels) of nucleotides at the site of damage [11]. This error-prone repair mechanism results in

frameshifts at the break site (Fig. 2.), and if the region encodes for a gene, its function is knocked out

[11]. On the other hand, HDR employs a template sequence that is homologous to the break site [11].

This high fidelity repair mechanism is mainly active during the S and G2 phases of mammalian cell

cycle, and the sister chromatid is used as template for the break sites [11]. However, an exogenous

template may be introduced to flank these break sites to incorporate synthetic sequences (Fig. 2) [11].

The CRISPR-Cas system has been praised for its efficiency, precision, low cost and ease of

use [8]. Furthermore, for multiplexing purposes, this system has the ability to simultaneously edit

multiple target sites without the need of cumbersome protein engineering as required in ZFNs or

TALENs [7][8]. Three types of CRISPR systems have been identified, however, only the type II

CRISPR-Cas system is the least complex as it only requires three components to function; Cas

endonuclease, mature crRNA and auxiliary tracrRNA [2][7]. This system has been further simplified

by fusing the crRNA with the tracrRNA to form a single guide RNA (sgRNA) [7][11].

Figure 2: Genome double strand break (DSB) repair mechanism. Single endonuclease-induced DSB (left): Non-homologous end-joining

(NHEJ) knocks out gene function by altering the gene’s original stop codon location. Homology-directed repair (HDR) occurs via strand

invasion into complementary templates. HDR may knock-in point mutations when provided with a mutated exogenous flanking template

(light blue dsDNA with star). HDR may knock-in new gene function when the exogenous flanking template contains its coding sequence

(light blue dsDNA with orange regions). Dual endonuclease-induced DSB (right): NHEJ large sequence deletions. (Figure adapted from

[11])

The type II CRISPR-Cas9 system is derived from Streptococcus pyogenes, and this powerful

genome editing tool has already been employed to alter the genes of different organisms and cell

types ranging from microorganisms, insects, plants, fishes, to mammalian cell lines [12]. Here, I will

review a few CRISPR-Cas9 genome editing applications, limitations and optimizations.

2. Applications

2.1. Disease Modeling

Mice are routinely used to study mammalian genetics and associated diseases by knocking

out or knocking in specific genes through embryonic stem cell (ES) homologous DNA recombination

methods [13]. As these methods were first established in mouse ES cells, genome editing has been

limited to mice. This posed difficulties in editing other mammalian species that may be better suited

to model unique human diseases [13][14]. Furthermore, it may take up to a year or more to

successfully produce a genetically altered mouse [8].

In human cell lines, homologous recombination methods have had little rate of success such

that alternative approaches with short interfering RNA have become common [8]. These alternatives

also bring about their own set of limitations such as transient gene expression knock-downs and off

target effects contributing to experimental inaccuracies [8].

Recent developments in genome editing technologies have enabled researchers to circumvent

the limitations around animal and cell line based disease modeling. Advantages include the ability to

genetically tweak animals for which ES cell lines are unavailable, the potential to acquire

homozygous knock-outs within the first generation, and the opportunity to explore other types of

animal models [8].

With CRISPR-Cas9 multiplexed genome editing, multiple generations of interbreeding to

derive an animal model with many genetic modifications can be avoided. This multiplexing potential

has been demonstrated to knock-out two genes of a monkey in one step by co-injecting one-cell-stage

embryos with Cas9 mRNA and sgRNAs [15]. In mice, CRISPR-Cas9 multiplexed genome editing of

two genes produced biallelic mutations with eighty percent efficiency [16]. Also, the CRISPR-Cas9

system empowers researchers to study single nucleotide polymorphism-associated human diseases

efficiently in mice by introducing a donor template that encodes the mutated sequence (Fig. 2) [14].

These remarkable achievements suggest that there may be no technical barrier in using the CRISPR-

Cas9 system to model other animals or cell lines for the development of new pharmaceuticals.

2.2. Drug Development

Genome-wide association studies, a field of study in functional genomics, have provided us a

wealth of knowledge on polygenic diseases such as Alzheimer’s, schizophrenia, autism and diabetes

[7]. Drug development for these genetic diseases relies heavily on disease model studies in zebrafish,

drosophila, mice and even mammalian cell lines using RNA interference (RNAi) [17][18]. As

mentioned previously in the disease modeling section, RNAi techniques have the tendency for partial

knock-downs and unmeasurable phenotypes [17]. Despite these limitations, genome-wide screening

for novel therapeutic targets have been using subsets of the RNAi technology, specifically, short

hairpin RNA (shRNA) and small interfering RNA [17].

The advancement of programmable genome editing platforms has enabled the generation of

gene knock-outs and mutation knock-ins in clinically relevant animal and cell based models for target

validation and drug discovery [17]. Unlike RNAi, which silences a particular gene expression by

targeting its mRNA transcripts, the CRISPR-Cas system targets its DNA coding sequence directly,

leading to permanent and complete gene knock-out [18]. As amendments made to the genome via the

CRISPR-Cas9 system are irreversible, alternative methods have been explored to mimic the RNAi

gene silencing approach, but at the DNA level as it confers greater efficiency [18]. A team of

scientists managed to create a dead Cas9 (dCas9) by inactivating its DSB catalytic mechanism [19].

Therefore, when guided by an engineered sgRNA, the dCas9-sgRNA complex interferes with the

targeted gene’s transcription, strongly silencing its gene expression [19]. This CRISPR interference

(CRISPRi) method appears to be most analogous to the RNAi principle.

In summary, the CRISPR-Cas9 system has enabled the study of other clinically relevant

animal models. Also, the ability to perform multiplexed genome editing and interference has opened

up greater opportunities for novel therapeutic target screening. Overall, functional genomics has

uncharted territories for us to explore in terms of polygenic disease knowledge and novel drug

development.

2.3. Regenerative Medicine

Many genetic disorders require individuals to be placed under prolonged or lifelong

medication. Based on current developments in genetic engineering, gene therapy may soon become a

primary route of treatment for genetic diseases [7]. Several proof of concept studies have

demonstrated the potential of gene therapy to correct monogenic recessive disorders such as

hemophilia, cystic fibrosis and Duchenne muscular dystrophy (DMD) [7][11]. On the other hand,

non-genetic diseases may also be prevented by modifying one’s genome.

Knock-out gene therapy relies on the error-prone NHEJ repair mechanism to induce indels at

the targeted gene, causing a frameshift mutation (Fig. 2) [11]. Familial hypercholesterolemia is an

autosomal dominant genetic disease caused by a mutation in the PCSK9 gene [20]. This gene codes

for a proteinase that degrades low density lipoprotein receptors (LDLR) and therefore contributes to

decreased metabolism of LDL cholesterol by the liver, increasing the risk for cardiovascular diseases

[20]. Patients with this condition are continuously prescribed PCSK9 proteinase-antagonists, however,

a single co-injection of Cas9 together with PCSK9-targeted sgRNA into mice liver in vivo has shown

to knock-out the targeted gene and effectively lower cholesterol levels [21]. Another promising

application of knock-out gene therapy is by disrupting the CCR5 gene encoding a major co-receptor

essential for HIV-1 to infect CD4+ T-cells [7][11]. Successful clinical trials have been observed with

ZFN edited stem cells being reintroduced into patients [7]. Similarly, engrafting Cas9 edited human

hematopoietic stem and progenitor cells appears to be a powerful alternative to combat AIDS [22].

Knock-in gene therapy employs the high fidelity HDR mechanism of cells to rectify

mutations by providing an exogenous template encoding the correct gene sequence to flank the DSB

site (Fig. 2) [11]. In addition, an alternative error-free insertion of exogenous genetic elements up to

fifteen kilobases have been developed using NHEJ-mediated ligation [23]. This is achieved by using

nuclease-induced DSBs to generate compatible overhangs on both the exogenous template and the

endogenous target site [23]. Both methods are advantageous compared to conventional viral vector-

mediated gene therapy associated with random insertion mutagenesis [11].

In certain cases, where knock-out or knock-in gene therapies may be inappropriate to rectify a

particular genetic disease, deletion-based gene therapy may be explored. To delete large genetic

elements several megabases in size from the genome, two targeted DSBs flanking the region of

interest must be simultaneously administered (Fig. 2) [11]. Using the CRISPR-Cas9 platform, this

method has been proven to be helpful in treating hemoglobinopathis by removing an entire BCL11A

erythroid-specific enhancer region [24]. Additionally, in DMD, where internal gene deletions lead to

frameshifts and as a result causes protein dysfunction, deliberate targeted excision of its exons can

rectify the frameshifts to generate a truncated, partially functional protein [11].

Targeted gene therapy with the CRISPR-Cas9 system hold much promise in the upcoming

decade as numerous proof of concept studies have already been established [7][11], and it is only a

matter of time before a flood of new clinical trials using this system will be approved.

2.3. Medical Microbiology

The abuse of antibiotics in medicine and animal agriculture has led to great selection pressure

on bacteria such that multidrug-resistant strains, especially the human pathogens, are of growing

concern as our pre-existing arsenal of useful antibiotics is shrinking. Another issue is that these

antibiotics have broad-spectrum targets and does not discriminate pathogens from beneficial normal

flora in our systems. Alternative novel antimicrobials have been explored using the CRISPR-Cas9

system to circumvent these limitations [25].

The sgRNA in the CRISPR-Cas9 system can be engineered to target virtually any essential

genomic regions of the bacteria for killing. As for the mode of delivery, preliminary studies employed

bacteriophages with phagemids encoding the Streptococcus pyogenes Cas9, the engineered crRNA

and the auxiliary tracrRNA [26][27]. These studies demonstrated effective killing of pathogenic

Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae and enterohemorrhagic Escherichia

coli by targeting their virulence genes [26][27]. Specificity was also observed in one of the study as

the sequence-specific Cas9 discriminatively targeted virulent from avirulent Staphylococcus aureus

[26].

The CRISPR-Cas9 system can also be re-programmed to target antibiotic resistance genes

residing within the bacterial genome or in plasmids [25]. This could possibly be a method to restrict

plasmid-borne antibiotic resistance between clinically relevant pathogenic strains. Also, re-sensitizing

pathogenic strains to previously obsolete antibiotics can re-expand our antimicrobial arsenal.

In summary, sequence-specific antimicrobials may be used to treat multidrug-resistant

infections without killing beneficial normal flora by either directly via genome disruption or indirectly

through re-sensitization to antibiotics. However, as with any antimicrobials, further studies have to be

conducted to evaluate the types of evolution this method could bring about due to selection pressure.

2.5. Industrial Microbiology

A number of industrially relevant fungi and yeast strains utilized for the production of biofuel

are tough to engineer because of their intricate genomes [28]. Saccharomyces cerevisiae, one of the

most commonly exploited microbial cell factories, have diploid or polyploidy stains that make

genome editing a tricky task. Conventional methods employ selection of markers that are co-

integrated into its genome for gene mutation, deletion and integration [28]. Metabolic engineering has

found the CRISPR-Cas9 system to be a versatile genome editing tool to overcome these types of

challenges [28].

Genome engineering of several different industrially relevant Saccharomyces cerevisiae

strains using the CRISPR-Cas9 platform has demonstrated successful biallelic disruption of a gene

with efficiency up to seventy-eight percent [29]. In addition, by harnessing the multiplex nature of the

CRISPR-Cas9 platform, genome engineering of up to five different genomic loci in Saccharomyces

cerevisiae has demonstrated to increase mevalonate titers up to forty-fold compared to wild-type

strains, although no overexpression of genes was done in the mevalonate pathway [30]. Therefore,

CRISPER-Cas9 can be used in strain optimization to yield greater amounts of important secondary

metabolites.

2.6. Agricultural Biotechnology

Genetically modified crops incorporated with transgene via the HDR mechanism undergoes

strict regulations, decreasing their commercial viability [7]. NHEJ knock-outs however, are said to be

non-transgenic and is beyond the United States Department of Agriculture (USDA) regulatory

authority [31]. Highlighted here are a few prominent CRISPR-Cas9 based crop and livestock genome

editing that have taken place over the past few years.

Engineering plants and crops to be resistant to diseases is one of the major goals for

agricultural biologists. Leveraging upon the superior gene silencing and multiplexing capabilities of

the CRISPR-Cas9 system over RNAi, a number of model plant and crop species have been further

studied [7]. Examples include Arabidopsis, tobacco, tomato, maize, rice and wheat [7]. The powdery

mildew disease is a deadly wheat infection caused by the fungus Blumeria graminis [7]. In bread

wheat, Triticum aestivum, a hexaploid crop, three mildew resistance locus (MLO) homoeoalleles were

mutated via CRISPR-Cas9 [32]. Subsequent self-fertilization yielded knock-outs for all six alleles,

conferring the bead wheat resistance to powdery mildew disease [32].

Biofortification is the process by which plants, crops and livestock are genetically engineered

to enhance their nutritional value. Improvements can be made directly by modulating the amount of

nutrient the foodstuff produces, or indirectly by eliminating anti-nutrients that decreases nutrient

bioavailability [7]. Goat, sheep, pig and cattle have already been successfully engineered using the

CRISPR-Cas9 system [7]. Other biofortification strategies using CRISPR-Cas9 are still under

development, and although ZNFs and TALENs have already been extensively used, the potential of

CRISPR technology to improve the nature of agricultural produce and livestock remains massive.

3. Limitations

3.1. Target Specificity

Off-targeting, whereby DSBs are made at sites other than the intended target region, has been

one of the greatest limitation of not only the CRISPR-Cas9 system, but also other genome editing

tools within the family of programmable nucleases [2][8]. Should off-target mutations occur

frequently using the CRISPR-Cas9 system, there may be a loss of genomic stability and functionality

of otherwise normal genes, reducing the reliability of the tool for biomedical and clinical applications.

The targeting specificity of Streptococcus pyogenes Cas9 is tightly controlled by the twenty-

nucleotide guide sequence in the sgRNA and the PAM (typically NGG) sequence located beside the

target region (Fig. 3) [2][8]. However, potential off-target DSBs could be induced if there is three to

five base pair mismatch in the PAM-distal region of the guide sequence (seed region) [2].

A genome-wide binding analysis in mouse embryonic stem cells using dCas9 and chromatin

immunoprecipitation followed by sequencing (ChIP-seq) revealed a distinct seed region for on-target

binding, but also multiple off-target binding sites [2]. However, majority of these off-target regions

were not cleaved by catalytically active Cas9 [2].

Based on the catalytic activity, exceptionally high or low GC content within the sgRNA could

cause the guided Cas9 to be less active [33]. Also, methylation of DNA at CpG sites have been

reported to decrease Cas9 binding efficiency [2].

Figure 3: Cas9 endonuclease guided by sgRNA to target sequence. Cas9 endonuclease (large green structure) containing the sgRNA

(small green loop structure) is shown interrogating the target DNA (blue double strand structure). Scissors indicate Cas9 cleavage site three

base pair upstream the PAM sequence. The single letter DNA code depicted encodes for (N=A, T, C, G; R=G or A). (Figure adapted from

[2])

3.2. Delivery Techniques

Lentiviral and Adeno-associated virus (AAV) vectors have been commonly used to transfer

genetic information into cells. However, they do not have sufficient capacity to shuttle an entire Cas9

genome editing infrastructure [7]. Although other viral vectors such as adenovirus possess greater

carrying-capacity, they are highly immunogenic and have limited cell-type infectivity [7].

4. Optimizations

4.1. System Modifications

Three different forms of modification have been made to the Cas9 system to increase

targeting fidelity. Firstly, inactivation of the RuvC nuclease domain generates a Cas9 with nickase

(nCas9) functionality, causing only targeted single strand breaks in DNA instead of DSBs [7].

Appropriately spaced nicks formed with two nCas9 can mimic the effects of DSB, reducing the off-

target frequency as compared to using only one endonuclease [7].

Secondly, as off-target DSBs are mainly due to the strong binding affinity between the

sgRNA and non-specific target site, a truncated guide sequence could reduce the binding affinity such

that mismatched base pairs are no longer well tolerated, decreasing off-target editing [7][8].

Lastly, dCas9 can be engineered with Fok I nuclease domain to generate fCas9 [7]. A paired

fCas9 functions analogously to the paired nCas9, and has up to 140-fold increase in specificity

compared to wild type Cas9 [7].

Earlier this year, researchers have reported the generation of a high-fidelity CRISPR-Cas9

system capable of precise genome edition with undetectable off-target genome disruption [36].

4.2. Alternative Delivery Techniques

To overcome the capacity limitations of viral vectors, the essential Cas9 genome editing

modules have been engineered into separate plasmids and co-transfected into cells [7]. For in vivo

disease modeling, mice have been engineered to express Cas9 in a Cre-driven manner, such that only

the sgRNA is required for efficient genome editing [7]. Another novel method to introduce Cas9

proteins into cells involve fusion with anionic supercharged GFP that are delivered to human cell

lines and mice using cationic liposomes [35].

Direct introduction of purified Cas9 proteins together with the engineered sgRNA into cells

have shown to minimize off-target DSBs compared to plasmid-based Cas9-sgRNA expression [34].

The increased fidelity was reported to be derived from the rapid degradation of these Cas9-sgRNA

complexes in cells [34].

5. Conclusion

In recent years, research and application of the CRISPR-Cas9 system have picked up pace.

The advantages associated with this system compared to other programmable nucleases are

efficiency, specificity, time and cost saving. Furthermore, its ability to conduct reliable multiplexed

applications have opened up the opportunity for us to explore the vast knowledge of functional

genomics unattainable previously with RNAi technology.

This genome editing tool is providing advancement in many scientific fields. Enhancing

agriculture, defending against pathogens and remediating human genetics are just few of the benefits

listed from the large pool of published studies. With the recent development of high-fidelity Cas9 that

generates no detectable genome-wide off targets, many human-based clinical trials harnessing this

powerful genome editing platform are expected to be approved sooner than later.

Many questions still remain about how the innate immune system of organisms targeted with

CRISPR-Cas9 will respond. As of now, little is known on the selection pressure or evolution these

types of genome editing can bring about. Nevertheless, as many genome editing preclinical studies

and clinical trials have been successful, there is much hope and optimism for the future of genome

editing with CRISPR-Cas9 technology.

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