CRISPR-Cas9: Components and Application

Jiawen Jiang

Department of Molecular and Cell Biology, University of Connecticut, 82 N. Eagleville, Mansfield, U.S.A.

Keywords: CRISPR-Cas9 System, sgRNA, crRNAs, Gene Editing, DNA Endonuclease, RuvC, HNH.

Abstract: The identification of components of the CRISPR system discovered in bacterial cells enables gene editing in

a more efficient way. The further upgrading allows researchers to easily edit the DNA sequence not only in

prokaryotic cells but also in mammalian cells. The novelization of the CRISPR-Cas9 system promotes broader

utilization of this technique, resulting in staggered cuts in dsDNA sequences. Even during the COVID-19

pandemic, the employment of CRISPR-Cas9 techniques accelerates the development of SARS-CoV-2 testing

kits, allowing fast and effective testing available to a massive population. In this paper, I describe the progress

of finding the components followed by the novelization as well as applications of the CRISPR-Cas9 system.

1 INTRODUCTION

An impassioned discussion about the CRISPR

(Clustered Regularly Interspaced Short Palindromic

Repeat) system has been initiated because of the

Nobel Prize winner Emmanuelle Charpentier and

Jennifer Doudna, the pioneers of CRISPR

technology, in 2020 (Savić, Schwank 2016). Early in

1987 when CRISPR has been discovered for the first

time, a sequence located in the bacterial genome that

disarms bacteriophages infection by cutting off the

phage’s DNA (Doudna, Charpentier 2014). This

bacterial adaptive immune system was first being

used for genome editing in 2012 when Charpentier

and Doudna thoroughly discover the feature of the

CRISPR-Cas9 system which can recognize a specific

sequence of DNA and cut off the target site (Savić,

Schwank 2016). The nuclease Cas 9 followed by two

guide RNA sequences together constitute the genetic

scissor (Savić, Schwank 2016). Since then, they were

also able to target different DNA sequences by

reprograming the sequence of the short RNA

(Doudna, Charpentier 2014). Scientists are now able

to genetically modify the sequence of short RNA and

insert that RNA inside Cas 9 protein to target and

disable the desired sequence of DNA in cells (Zhang

et al. 2014). Engineering not only the sequence of the

guide RNA but also the protein Cas 9 enable scientists

to add, delete, disable, enhance, or even replace the

sequence of interest much more easily than other

https://orcid.org/ 0000-0002-5449-2632

genome editing techniques such as Zinc Finger

Nuclease (ZFN), and Transcription-Activator Like

Effector Nucleases (TALEN) (Gupta et al. 2019).

This precious research achievement brings not

only people in the science field, but ordinary people’s

attention towards the application, advantages,

challenges, and potential shortcomings of this

technique. Thousands of questions have been

delivered to scientists to answer but some of them

have not yet been responded systematically.

In this review, with the aim of collectively and

comprehensively discussing why and how the

CRISPR-Cas 9 system deserves this massive

attention, the content of introduction of the CRISPR-

Cas 9 system, potential applications of CRISPR

technology, the progress of cutting-edge technology,

and the future opportunities of CRISPR-Cas9 will be

included. The significance of the objective focuses on

providing a thorough introduction of the CRISPR-

Cas9 system as well as its up-to-date application to

promote learning, thinking, and potential

breakthrough within this field.

744

Jiang, J.

CRISPR-Cas9: Components and Application.

DOI: 10.5220/0011294300003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 744-748

ISBN: 978-989-758-595-1

2 CRISPR-Cas9 SYSTEM

2.1 The Understanding of

CRISPR-Cas9

CRISPR-Cas9 was first understood by Jennifer

Doudna and Emmanuelle Charpentier in 2012 (Jinek

et al. 2012). Before 2012, Dr. Doudna worked on

understanding and analyzing the composition of the

CRISPR-Cas9 system which has been found out to be

involved in the bacterial innate immune system to

defend against viral infection (Wiedenheft et al.

2009). Working along with Dr. Charpentier, Dr.

Doudna identified the inner component of Cas9

protein consisted in the type II CRISPR-Cas9 system

(Hryhorowicz et al. 2017).

Cas9 is a DNA endonuclease guided by two

RNAs, one is called CRISPR RNAs (crRNAs) and

the other is trans-activating crRNAs (tracrRNA)

(Jinek et al. 2012, Cong et al. 2013). crRNA is

complementary to the sequence that Cas9 is targeting,

while tracrRNA is essential to crRNA maturation as

well as triggering plasmid cleavage (Jinek et al. 2012).

A specific motif appears in the foreign genome called

protospacer adjacent motif (PAM), 2-6 nucleotides

downstream of the cut site, also need to be recognized

by crRNA, and cut by Cas9 3-4 nucleotide upstream

of it (Jiang, Doudna 2017). Once part of the sequence

is fully recognized, Cas9 protein bind onto the strand,

making the cut using two domains, each domain cut

one DNA strand (Jinek et al. 2012) (Fig. 1a). By

utilizing radioactivating tags on one of the DNA

strands, Doudna et al. determined that the HNH

domain is responsible for complementary strand

cleavage, while the RuvC-like domain is for the

uncomplimentary strand cleavage (Jinek et al. 2012).

A linear dsDNA is obtained after the cleavage

showing a successful cut by Cas9 (Jinek et al. 2012).

Although this is a part of the innate immune

system of multiple bacteria such as Streptococcus

pyogenes, the research found out that the CRISPR-

Cas9 system is programmable and can be used in

other organisms by changing the guide RNA (Chen et

al. 2019). Moreover, crRNA and tracrRNA can be

linked together with a hairpin to become a chimeric

RNA that mimics crRNA: tracrRNA complex (Chen

et al. 2019, Ran et al. 2013). Studies show that by

editing the sequence of chimeric RNA, Cas9 protein

with a single chimeric guide RNA is potentially

capable of cutting any dsDNA in many organisms,

resulting in a new era of genomic regulation and

genomic editing (Chen et al. 2019, Ran et al. 2013).

Figure 1. The CRISPR-Cas9 system. a. Double strand DNA breaks and binds to Cas9 and single guided RNA. Each domain

cut one strand of the DNA and resulting in double-strand break of genetic material (Chen et al. 2019). The original circular

DNA will become linear DNA due to the cut (Jinek et al. 2012). b. The double-strand lesions will be repaired through either

a non-homologous end joining or a homologous repair pathway. Both NHEJ and HDR allow the desired sequence to be

eliminated from the DNA achieving the goal of attacking foreign DNA (Chen et al. 2019, Ran et al. 2013, Cong et al. 2013).

2.2 Repurposing CRISPR-Cas9 System

The CRISPR-Cas9 system is programmed not only to

cut dsDNA but also to manipulate transcription in

eukaryotes (Qi et al. 2013). Inactive Cas9 or

denatured Cas9 (dCas9) protein is a great model in

the experiment (Cong et al. 2013) (Fig. 2). In this case,

the Cas9 lacking the endonucleolytic activity can

normally bind to complementary strand DNA but

cannot cut desired sequence [Qi et al. 2013]. Research

has shown that while Cas9 binds to the dsDNA, the

transcription is blocked with low off-target effects (Qi

al. 2013, Hsu et al. 2014). Furthermore, Cas9 has

CRISPR-Cas9: Components and Application

745

Figure 2. The programmable Cas9 protein and single-guide RNA (sgRNA) enable transcription regulation (Qi et al. 2013).

The wild type Cas9 protein cut results in the forming of linear DNA (left), while a dead Cas9 protein with a modified sgRNA

can bind onto the DNA sequence and shut down the original transcription by occupying the transcriptional site where the

RNA polymerase (RNAP) needs to bind (right) (Qi et al. 2013).

been used in multiple eukaryotic organisms to test the

efficiency and accuracy of genetic regulation and as a

result, the outcome is positive in most of the

organisms (Hsu et al. 2014).

The system originated from bacteria is used to cut

off and silence viral DNA (Jinek et al. 2012, Ran et

al. 2013). Silencing of the desired segment of dsDNA

in eukaryotic cells is achieved by cutting off the target

sequence followed by adding more nucleotide base

pairs to close the gap (Ran et al. 2013). Cas9 cutting

resulting in target double-strand DNA breaks induces

non-homologous end-joining or homologous repair

pathway (Chen et al. 2019, Cong et al. 2013, Hsu et

al. 2014) (Fig. 1b). Both pathways contribute to

precise gene editing by silencing a specific gene

transcription with very low off-target effects.

Furthermore, research carried out in mammalian cells

indicated that the CRISPR-Cas9 system could also

show high efficiency of creating target DNA lesions

with the desired sgRNA (Hsu et al. 2014).

Cong et al. contributed significantly to the

understanding and further investigation of the type II

CRISPR-Cas9 system in human cells (Cong et al.

2013). Human codon-optimized Streptococcus

pyogenes Cas9 (SpCas9) and RNase III (hSpRNase

III) are modified versions of Cas9 and RNase that can

be better used in human cells and at the same time

have less error with higher efficiency (Cong et al.

2013).

According to Cong et al., the type II CRISPR-

Cas9 system can be as efficient and accurate as of the

TALENs which has been used in genetic editing prior

to the discovery of CRISPR technology (Cong et al.

2013). Furthermore, they claimed that CRISPR-Cas9

system can do multiplexed genome engineering

(Cong et al. 2013). Cas9 is engineered to target both

EMX1- and PVALB loci. The efficiency is 1.6%,

indicating the ability to do multiplexed editing in a

single genome (Cong et al. 2013). The efficiency of

cutting is diminished by mismatches between the

sgRNA and target DNA (Slaymaker et al. 2015).

Neutralization of positively charged groove between

HNH, RuvC, and PAM interacting domains on the

nontargeting DNA strand promotes rehybridization

between dsDNA strands and hence encourages tight

binding between sgRNA and target DNA strand

(Slaymaker et al. 2015). This upgrade by Skaymaker

et al. allows CRISPR-Cas9 system to reach a newer

level with a very low off-target effect in terms of

target cutting (Cong et al. 2013, Slaymaker et al.

2015).

2.3 Novelizations and Progression of

the Application of CRISPR-Cas9

System

More modifications on the Cas9 system by Zetsche et

al. enabled more various binding and cutting. For

example, a novel version of Cas9 called Cpf1 and

C2c2 endonuclease achieved a staggered cut on

dsDNA (Zetsche et al. 2015).

Another significant update on CRISPR-Cas

system by Gootenberg et al. was using the method of

specific high-sensitivity enzymatic reporter

unlocking (SHERLOCK) to detect foreign strains in

small amounts such as the infection by Ziska and

Dengue virus (Gootenberg et al. 2017). The

advantage of this technique is that it can sense viral

and bacterial pathogens in samples that are relatively

advisable such as human serum or urine samples

(Gootenberg et al. 2017). Due to the high accuracy of

the technique, it can distinguish between similar viral

or bacterial strains successfully (Gootenberg et al.

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2017). It can also distinguish SNP in human

genotyping (Gootenberg et al. 2017).

During the COVID-19 pandemic, Joung et al.

discovered STOPCovid, a technique used to rapidly

detect the genetic material of SARS-CoV-2 from oral

or nasal swab samples giving either positive or

negative outcomes using detection strips or specific

values of viral RNA counts under fluorescence

readers (Joung et al. 2020). The overall sensitivity is

93.1% and specificity is 98.3% among 402 candidates

(Joung et al. 2020).

The rapid, inexpensive, and sensitive detection

enables CRISPR-Cas system to be highly used since

2015 in genetic regulation and genomic editing

(Gootenberg et al. 2017, Joung et al. 2020).

Recently, studies have shown that the success of

CRISPR-Cas9 technology inspired therapy in

Huntington’s disease, sickle cell diseases, cancer, and

B-thalassemia, indicating more potential treatments

available in the future targeting different diseases

(György 2021).

3 DISCUSSION

Owing to the concern of creating genetic mutation

due to permanent change in genes in human cells by

the CRISPR-Cas9 technology, it is better having

safely regulate the on-and off-switches of the

CRISPR-Cas9 system (Shivram et al. 2021). In case

of observing relatively high off-target effect that may

lead to serious genetic defects as well as

unpredictable diseases of an organism, having types

of “emergency shut down system” is necessary for

more complicated gene editing events, especially

when introducing DNA cut and gene segment

replacement in mammalian cells or even in human

cells. Regulators from Escherichia coli and

Salmonella typhi are identified as having H-NS and

LRP which are responsible for negative regulation of

Cas expression, while LeuO can positively regulate

Cas promoter to further simulate CRISPR-Cas cutting

(Shivram et al. 2021). Those genes, in theory, are

responsible for regulating Cas system, as well as

being potential switches for CRISPR-Cas9 system,

but the future investigation is needed to make sure

those factors found in bacteria can be equally

beneficial with high efficiency in mammalian cells.

Moreover, even though the off-target effect is

significantly lowered by Zhang et al. after several

steps of upgrading, it is not possible for now to achive

0 off-target effects and as a result, it is still unknown

if the efficiency can promote a positive outcome in

clinical trials (Cong et al. 2013, Zetsche et al. 2015).

4 CONCLUSION

The existence of components located in CRISPR-

Cas9 system allow further investigation and

advancement in other fields. The revelation of the

ability to practice precise cut by RuvC-like and HNH

domain, engineer sgRNA, and replicate the

mechanism in eucaryotic cells results in a higher

frequency of the usage of the CRISPR-Cas9

technique in labs regarding the aim of doing gene

editing. This technique is known as the most rapid

and the easiest method in genetic regulation and

genomic engineering. Not only the Noble Prize

winner Emmanuelle Charpentier and Jennifer

Doudna but also other scientists progressively and

largely contribute to the refinement of the CRISPR-

Cas9 system in their research fields. Based on all of

the existing gene editing products by the CRISPR-

Cas9 system, investigating an efficient way of fully

controlling the on and off of CRISPR-Cas9 is critial

as well as worth studying in order to make CRISPR-

Cas9 a precise gene editing machinery. This paper

about CRISPR-Cas9, an influential topic in the

science field, hopefully, will promote thinking as well

as more discoveries that may lead to better and novel

disease treatments in the future.

ACKNOWLEDGEMENT

I would like to extend my sincere thanks to Dr.

Gerwald Jogl at Brown University for providing

advice on how to improve this paper.

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