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공학석사 학위논문

The Effect of Transcriptional

Activators via a Blue Light-Inducible

CRISPR-Cas9 System

청색 광유도 유전자 교정 시스템에서의

전사인자 활성체의 영향

2020 년 2 월

서울대학교 대학원

협동과정 바이오엔지니어링 과정

Ninj Byambasuren

1

ABSTRACT

The Effect of Transcriptional

Activators via a Blue Light-Inducible

CRISPR-Cas9 System

Ninj Byambasuren

Interdisciplinary Program in Bioengineering

The Graduate School of Engineering

Seoul National University

Increasing interest in genetic engineering has warranted the need for highly dynamic

control of gene upregulation, which has resulted in improvements to the conventional

CRISPR activation system (CRISPRa). One of these improvements, the light-inducible

CRISPR activation system (LICAS) utilizes light-inducible heterodimers CIBN and

CRY2FL fused to catalytically deactivated Cas9 (dCas9) and a transcriptional activator

VP64. LICAS dynamically regulates endogenous gene expression in the presence of

blue light. Recently, researchers developed the tripartite transcriptional activator VPR

(VP64-p65-Rta), which showed higher levels of transcriptional gene activation than

VP64 when utilized for the conventional CRISPRa system. However, VPR’s activation

levels for LICAS are yet to be determined. Therefore, in this study, we investigated

2

VPR and VP64 in CRISPRa and LICAS to compare their gene activation levels. First,

we investigated dCas9-VPR and dCas9-VP64 in CRISPRa by targeting the Interleukin

1 Receptor Antagonist (IL1RN) gene. We confirmed previous studies contending that

VPR induced higher gene activation than VP64 for IL1RN. In LICAS, when we

compared VPR with VP64 by fusing each transcriptional activator to a full-length

cryptochrome (CRY2FL), higher gene activation was observed via CRY2FL-VP64.

Nevertheless, we demonstrated that our novel CRY2FL-VPR construct succeeded in

activating the IL1RN in LICAS. However, its activation efficiency was relatively weak

compared to CRY2FL-VP64. Despite this fact, we proved that VPR activator domains

can be fused to CRY2FL without deactivating its ability to complex and function with

sgRNAs in human cells. With further investigation, the CRY2FL-VPR design may

propel improvements to dynamic endogenous gene activation methods for genetic

research and therapy.

Keywords: CRISPR-Cas9, CRISPR-mediated activation system (CRISPRa), a

light-inducible CRISPR activation system (LICAS), VPR, VP64, CRY2FL-CIBN,

optogenetics

Student Number: 2018-24978

3

Table of Contents

ABSTRACT ................................................................................................................... 1

Table of Contents ............................................................................................................ 3

Chapter 1. Overview of CRISPR ................................................................................... 4

1.1 Improvements to the CRISPR-Cas9 System ................................................. 4

1.2 CRISPR-mediated Activation and Types of Transcriptional Activators ....... 6

1.3 Development of a Light Inducible CRISPRa System ................................... 7

Chapter 2. Developing a light-inducible CRISPR activation system utilizing CRY2FL-

VPR ................................................................................................................................ 9

1. Introduction .................................................................................................... 9

2. Materials and Methods ................................................................................... 12

2.1 Cell Culture ......................................................................................... 12

2.2 Transfection ........................................................................................ 12

2.3 Plasmid Construction and Quantity .................................................... 12

2.4 Endogenous gene targets and sgRNA design ..................................... 13

2.5 Quantitative real-time PCR analysis .................................................. 13

2.6 Blue light construction and illumination ............................................ 14

2.7 Statistical analysis ............................................................................... 14

3 Results ............................................................................................................. 15

3.1 Construction of endogenous gene activation system based on the

dCAS9 system. .............................................................................................. 15

3.2 Endogenous gene activation via dCas9-VPR and dCas9-VP64 in

CRISPRa. ....................................................................................................... 16

3.3 Design of light-inducible CRISPRa system (LICAS) based on

CRY2FL-VPR and CIBN-dCas9-CIBN ....................................................... 16

3.4 Activation of an exogenous eGFP reporter by CRY2FL-VPR. ......... 17

3.5 Comparison of CRY2FL-VPR to CRY2FL-VP64 in LICAS. ........... 18

4. Discussion ...................................................................................................... 18

Figures and Tables ................................................................................................. 22

References .................................................................................................................... 30

4

Chapter 1. Overview of CRISPR

1.1 Improvements to the CRISPR-Cas9 System

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are a

family of DNA sequences that work as a bacteriophage defense mechanism in bacteria

and archaea [1]. The CRISPR and CRISPR associated (Cas) proteins serve together as

a form of genetic memory that help the bacterial cell detect and destroy harmful viral

invaders [2]. Cas proteins are a useful tool for molecular testing due to their ability to

specifically target DNA sequences. The CRISPR system and Cas proteins form the

foundation for the development of CRISPR based genome editing tools, such as

CRISPR-Cas9 [3]. Compared to other Cas proteins, Cas9 is most commonly used

because it is the first-identified and best-characterized single-protein CRISPR activator

[5]. There are two main components of the CRISPR-Cas9 system: a single guide RNA

(sgRNA) that identifies a protospacer adjacent motif (PAM) sequence and immediately

follows the targeted DNA sequence and the Cas9 protein which produces double-strand

breaks (DSBs) at the target site that are later repaired by cellular mechanisms [4, 5].

In 2013, researchers repurposed Cas9 to target DNA without cleaving it, termed

nuclease-deactivated Cas9 or dead Cas9 (dCas9) [5]. Cas9 has two catalytic domains

(HNH and RuvC) that act together to mediate DNA double-strand breaks (DSBs).

Endonuclease activity is removed through a single point mutation in either of these

domains, which results in a nickase enzyme that only cleaves one side of the target site.

Mutations in both domains (D10A and H840A for SpCas9) result in complete loss of

DNA cleavage activity [4]. By modifying Cas9 into dCas9, researchers now have the

5

ability to regulate gene expression, perform epigenetic manipulation, and CRISPR

imaging [8, 10-12] (Fig.1).

The CRISPR activation system (CRISPRa) utilizes dCas9 fused to

transcriptional activator domains (e.g. VP64, P65, Rta5) to upregulate gene expression

[6]. Conventional approaches introduce exogenous genes into cells to promote targeted

gene expression (e.g. plasmid with a CMV promoter, DOX inducible gene activation,

artificial transcription factors) [7, 8]. However, for the first time, CRISPRa allowed for

overexpression of genes in their endogenous context and enabled targeted

transcriptional activation [9]. Yet, to precisely control gene expression, highly dynamic

and inducible control of CRISPR-dCas9 was sought after [10]. Researchers have

developed systems that enable precise control and inducibility of CRISPR-dCas9

through the utilization of diverse tools: small molecules [11], ligands [12], a split-Cas9

[13], and chemicals (e.g. doxycycline-inducible sgRNA) [8]. However, these methods

have several adverse effects because their byproducts diffuse freely and have

unintended effects, making it difficult to achieve highly inducible genome editing

without drawbacks [14]. A solution for these limitations may be a light-inducible system.

A light inducible system has high spatiotemporal resolution and is noninvasive

compared to other inducible methods [15].

Therefore, in 2015, the blue light-inducible CRISPR-Cas9 activation system

(LICAS) was developed to achieve repeatable and reversible control of endogenous

gene expression [10]. LICAS induces gene activation by utilizing blue light-dependent

dimerization proteins: a full-length cryptochrome (CRY2FL) and its binding protein

(CIBN) from Arabidopsis thaliana. CRY2FL was fused to the transcriptional activator

domain VP64 resulting in a CRY2FL-VP64 construct. CIBN was fused to the N- and

C-terminus of dCas9 resulting in a CIBN-dCas9-CIBN construct. In the presence of

6

blue light, CRY2FL binds to CIBN, which recruits and transports CRY2FL-VP64 to the

target locus and initiates gene expression [16]. The single-domain transcriptional

activator VP64 is an essential component of the currently-used LICA system.

Incidentally, the tripartite activator VPR (VP64-p65-Rta) has shown higher activation

efficiency than VP64 when utilized for the conventional CRISPRa system, the

precursor system to LICAS. In recent literature, when compared to dCas9-VP64,

dCas9-VPR showed ~22 to ~320 fold increased activation of endogenous targets [6].

Hence, in this study, we constructed a novel CRY2FL-VPR plasmid and CIBN-dCas9-

CIBN plasmid to investigate VPR and VP64’s relative efficiencies in LICAS.

1.2 CRISPR-mediated Activation and Types of

Transcriptional Activators

The dCas9-based CRISPR system has been adapted to generate tools called

CRISPRi (CRISPR interference) and CRISPRa (CRISPR activation) [17]. These tools

utilize nuclease-deactivated Cas9 (dCas9) that can't generate DSBs to target genomic

regions, resulting in RNA-directed transcriptional control. In CRISPRa, the dCas9

protein was engineered to be fused to a transcriptional activator (e.g., VPR, VP64) that

can be directed to promoter regions by sgRNAs to upregulate expression of the

endogenous gene target [18]. Examples of these systems are as follows: dCas9 fused to

artificial transcriptional activators (e.g., VP64, P65), dCas9 fused to an activator while

a tagged gRNA recruits other activators (SAM) [19], and a scaffold that recruits

activator peptides (SunTag) [20].

In recent years, to enhance transcriptional activation dCas9 has been fused to

multiple activator domains (VPR) rather than single activator domains (Fig 2. A).

7

Although the fusion of dCas9 to stronger activators results in higher levels of gene

activation, for dynamic control of gene activation, an inducible CRISPR-dCas9 system

is still necessary. Researchers have already developed systems for precise control of

CRISPR-Cas9 using small molecules [21], ligands [12], a split-Cas9 [13], chemicals

(e.g. doxycycline-inducible sgRNA) [8], and optics (e.g. LACE) [10]. However,

some chemicals, small molecules, and ligands have adverse effects because they diffuse

freely and are difficult to rapidly remove, which makes it hard to achieve spatiotemporal

genome editing [22]. Because light has high spatiotemporal resolution and is

noninvasive [23] we focused on a light-inducible CRISPR activation system.

1.3 Development of a Light Inducible CRISPRa System

While chemical-based inducible methods of Cas9 have been widely exploited,

the literature on light-inducible Cas9 methods are few. In contrast to chemicals, light is

a more ideal inducer because of its high spatiotemporal resolution and noninvasiveness

[24]. Researchers have developed several optogenetic transcriptional control systems

for dynamically regulating gene expression with light [23, 25, 26]. Most of these

systems use light-inducible heterodimerizing proteins (light-, oxygen- or voltage-

sensing (LOV) proteins, blue light–utilizing Flavin (BLUF) proteins, a light sensitive

cryptochrome (CRY2) and plant phytochromes (PHY)) from plants [24]. In application,

a heterodimerizing protein is fused to ZFP18 or TALE4 and its binding partner is fused

to a transcriptional activation domain (e.g., VP64). This enables control of any gene in

a reversible and spatiotemporal manner. However, reengineering the ZFP18 or TALE4

DNA-binding domain to target new sequences is difficult due to their complex

molecular structure [10]. To address these limitations, researchers adapted the CRISPR-

8

Cas9 activator system for optogenetic inducibility. A light-activated CRISPR/Cas9

effector (LACE) system was developed in 2015 to induce the transcription of

endogenous genes in the presence of blue light [27]. The light inducible

heterodimerizing proteins cryptochrome (CRY2) and the calcium- and integrin-binding

protein (CIB1) are fused to the transactivation domains, VP64 and dCas9, respectively.

Upon blue light irradiation, CRYFL2 and CIB1 heterodimerize and the transcriptional

activator, VP64, is recruited to the target locus activating gene expression. Therefore,

blue light can be used to spatiotemporally activate the CRISPR/Cas9 system and induce

gene activation.

9

Chapter 2.

Developing a light-inducible CRISPR

activation system utilizing CRY2FL-VPR

1. Introduction

Gene regulation has played a significant role in the development of cell and

tissue engineering [15]. Evolutionary factors such as homeostasis, environmental

adaptation, morphogenesis, disease regulation, and others are all maintained and

modified by gene regulation. Although genetic information is vital to cell development,

not every gene is actively produced. Genetic information has relatively high noise and

cells dynamically respond to environmental stimuli and cell events to meet the cell’s

needs. For example, in eukaryotes, cell to cell differences are a result of the expression

of different, specific genes. Further observation of gene functions in cellular processes

supports the need for approaches that enable spatial, temporal, and precise modulation

of gene expression. Simply put, gene editing itself is not enough for therapeutic or

engineering advancement. Dynamic regulation of gene expression is essential.

However, each dynamic gene regulation system has its drawbacks.

Regulatable transgene systems providing easily controlled, conditional stimulation or

repression of gene expression have already been developed for eukaryotes and are

crucial tools in biomedical research. Most of these systems depend on the

administration of either exogenous chemicals or heat shock. Despite the overall success

of the many of those systems, the potential for problems, like toxic, unintended, or

pleiotropic effects of the inducing chemical or treatment, can impose limitations on their

use. Therefore, highly specific noninvasive methods of gene regulation are still being

10

developed. Furthermore, the ability to manipulate the expression of endogenous genes,

as opposed to exogenous genes, has wide-ranging applications in medicine and applied

biology. With the advancement of dCas9 based tools, CRISPR-based editing has gained

interest in the genetic engineering field [18]. The development of CRISPRa enables the

upregulation of endogenous target genes without introducing exogenous genes into

cells [20]. Conventional approaches introduce exogenous genes into cells to enhance

specific gene expression (e.g. plasmid with a CMV promoter, DOX inducible gene

activation, artificial transcription factors) [13-15]. Other previously developed methods

upregulate gene expression through the addition of exogenous cDNAs into cells [19].

However, for the first time, CRISPRa allowed for the overexpression of genes in their

endogenous context and enabled targeted transcriptional activation [16] (Fig. 2A).

Furthermore, combining CRISPRa with optogenetics provides inducible and repeatable

control of gene activation [21].

Therefore, we chose to further develop a promoter system that can be induced,

rapidly and reversibly, by short pulses of light to specifically manipulate the expression

of endogenous genes known as the light inducible CRISPR activation system (LICAS)

(Fig. 2 B). With LICAS, we precisely control gene expression through highly dynamic

and inducible control of CRISPR-dCas9. Similarly, researchers have developed systems

that enable precise control and inducibility of CRISPR-dCas9 through the utilization of

diverse tools: small molecules [14], ligands [19], a split-Cas9 [20], chemicals (e.g.

doxycycline-inducible sgRNA) [21], and optics (e.g. LACE) [13]. However, some of

these chemicals, small molecules, and ligands have adverse effects because they diffuse

freely and are difficult to remove, as such it is difficult to achieve highly inducible

genome editing without drawbacks [22, 23]. A solution for these limitations can be

found in LICAS because it has high spatiotemporal resolution and is noninvasive

11

compared to other inducible methods [24]. LICAS is especially useful for dynamic

control of groups of genes and has potential applications in the clinical field of gene

therapy [22].

LICAS facilitates dynamic control of endogenous expression genes and may

be clinically applied to induce cell differentiation. The LICAS system activates genes

in the presence of gene-specific guide RNAs (gRNAs) and blue light. The system is

based on the plant proteins CRY2FL and CIBN from Arabidopsis thaliana that

heterodimerize in response to blue light. In LICAS, a full-length CRY2 is fused to the

transcriptional activator VP64 and an CIBN is fused to the N- and C-terminus of the

nuclease-deficient Cas9 (CIBN-dCas9-CIBN). In the presence of blue light, CRY2FL

binds to CIBN, which transports the CRY2FL-VPR complex to the gene target and

starts transcription.

In our study, light-dependent activation of the IL1RN and eGFP genes were

achieved by LICAS. We chose IL1RN our target gene because it is the most commonly

targeted gene in CRISPR editing due to its high levels of activation. Furthermore, the

IL1RN can be efficiently targeted with minimal risk of off-target Cas9 binding

elsewhere in the genome. The demand for a dynamic, noninvasive, efficient

endogenous gene regulation system is still an ongoing process and CRISPRa systems

provide a potential solution. Our choice to enhance LICAS was made to meet this

demand. We believe that our novel construction of CRY2FL-VPR and our efficiency

comparison of VPR and VP64 in LICAS both contribute to achieving a more holistic

and powerful gene activation system in the future.

12

2. Materials and Methods

2.1 Cell Culture

HEK-293T cell line cultured on 0.1% gelatin-coated culture dishes was purchased from

ATCC and maintained in a growth medium which consists of high glucose D-

MEM(Corning®) containing 10% FBS(Corning®), 1% Penicillin and

Streptomycin(Gibco™), and 1% Gibco® GlutaMAX™ supplement.

2.2 Transfection

For transfection, HEK293T cells at a density of 1.8×105 cells per well were grown

overnight in a 24-well plate and transfected with Neon Electroporation the next day

according to the manufacturer’s protocol. For CRISPRa cell line, 0.8M cells were

resuspended in 120ul resuspension buffer each containing 2ug of dCas9-VPR, dCas9-

VP64, and 2ug total sgRNA plasmids. For LICAS cell line, cells were transfected with

2ug of CRY2FL-VP64, CRY2FL-VPR, CIBN-dCas9-CIBN and sgRNAs plasmids.

The electroporation condition was optimized and the condition parameters are shown

in Table 1.

2.3 Plasmid Construction and Quantity

All plasmids described in this study: CIBN-dCas9-CIBN, dCas9-VPR, CRY2FL-VP64,

and empty backbone were obtained from Addgene (plasmid 60553, 99373, 60554, and

73311). The plasmid amounts for CIBN-dCas9-CIBN : CRY2FL-VP64 : minimal

eGFP reporter : sgRNA were 500ng : 500ng : 250ng : 500ng in a 24 well-plate with a

seeding density of 20,000 cells per well. For LICAS, the CRY2FL-VPR and CIBN-

13

dCas9-CIBN plasmids were cloned by Gibson Assembly. The eGFP reporter construct

was referenced from Addgene (plasmid 60718) and contains eight repeats of the

sequence 5′-AAAGGTCGAGAAACTGCAAA-3′ upstream of a minimal CMV

promoter and the gene encodeng eGFP [14].

2.4 Endogenous gene targets and sgRNA design

Several online tools (e.g., CRISPR Design or CHOPCHOP) were used to detect PAM

sequences and list possible sRNA sequences for IL1RN’s target region. We constructed

four sgRNAs for the IL1RN and their sequences are listed in the results section. The

sgRNA targeting promoter region of the eGFP reporter gene was 5’-

AAAGGTCGAGAAACTGGAA-3’. Four gRNAs were used together based on the

previous observation that multiple gRNAs are necessary to robustly activate gene

expression with constitutive dCas9-VP64 [a light].

2.5 Quantitative real-time PCR analysis

For quantitative real-time PCR analysis (qRT-PCR), cells were illuminated for 2 days

and the total RNA was extracted with 200ul of the TRIzol™ reagent (Invitrogen). 1ug

of total RNA was converted to cDNA in 20ul reaction volumes by cDNA samples that

were diluted by 5x and 3ul of the diluted cDNA (30ng or 6ng) was deposited in each

well of a 96-well Reaction Plate (Applied Biosystems). The qRT-PCR analysis was

performed with custom primers that are listed in Table 2. To detect target cDNA

amounts, SYBR Green with low ROX (Enzynomics) served as fluorescence signals.

Relative mRNA expression was determined by ΔΔCt method with glyceraldehyde 3-

phosphate dehydrogenase (GAPDH) as an endogenous control. Reactions were run on

a StepOnePlus Real-Time PCR System (Applied Biosystems).

14

2.6 Blue light construction and illumination

Cells were illuminated using a custom-built 4 × 6 blue LED array (~16 mW/cm2) with

450 nm, 48 lumens at 700 mA (Fig. 3). The blue light irritation (mW) was controlled

by the Ardiuno computer system and pulsed at an interval of 15 seconds. The current

was regulated using a BuckPuck AC Driver [14]. The LED array was held 20.32cm

above the incubator shelf with illumination facing downward such that the cells were

illuminated from the top of the dish. Because the plasmid construct contained an

enhanced fluorescence marker of green fluorescent protein (eGFP), after the irritation

period, cells in culture were visualized under a fluorescence microscope. Its area was

measured by ImageJ (NIH) analysis.

2.7 Statistical analysis

Data are shown as mean ± s.e.d. qRT-PCR data combined triplicates from one

independent experiment. Differentiation experiments were replicated in two

independent experiments with triplicate samples. Two-tailed Students t-test was

performed to explain whether two groups are from the same population or not with

statistical significance (P values). Statistical and graphical analysis was performed with

Prism (GraphPad) software. Asterisks were used to indicate statistical significance

between the two groups. P-values of less than 0.05 were considered significant (*P <

0.05, **P < 0.01, ***P < 0.001).

15

3 Results

3.1 Construction of endogenous gene activation system based

on the dCAS9 system.

Endogenous gene activation with the CRISPRa system requires either the

transcriptional activator VP64 or VPR, a catalytically deactivated Cas9 (dCas9), and

sgRNAs. In prior research, a SP-dCas9 fused with VP64-p65-Rta (VPR) or VP64 has

been constructed and utilized to activate endogenous genes. For this prior system, SP-

dCas9 is fused to VPR or VP64 at its C-terminus through the Gateway Cloning method.

In our study, we assayed the activity of both dCas9-VPR and dCas9-VP64 for their

inducible activation of the IL1RN endogenous gene in HEK293T cells. First, we

referenced the pcDNA-dCas9-VP64 (Plasmid #47107) and SP-dCas9-VPR (Plasmid

#63798) plasmids from Addgene. Then, we selected IL1RN-specific sgRNA targets

used in previous studies. We constructed these sgRNAs to localize dCas9-VPR and

dCas-VP64 to the promoter region of IL1RN. For IL1RN endogenous gene activation,

we utilized the genomic sequence of IL1RN (ENSG00000136689) and selected binding

regions for sgRNAs that were between 1 and 300bp upstream of the transcriptional start

site (TSS). Genome data was obtained from Ensembl Genome Browser.

We assayed the efficiency of dCas9-VPR mediated gene activation of endogenous

IL1RN by transfecting dCas9-VPR64 or dCas9-VPR and sgRNAs targeting IL1RN into

human embryonic kidney (HEK293T) cells.

16

3.2 Endogenous gene activation via dCas9-VPR and dCas9-

VP64 in CRISPRa.

To induce endogenous gene activation via dCas9-VPR and dCas9-VP64 in CRISPRa,

we transfected the HEK-293T cells with the necessary plasmid (500 ng) and sgRNAs

(500 ng). HEK293T cells at 70-80% confluency were transfected with Neon

Electroporation and seeded into a well of a 24-well plate. After a 48hr incubation, the

cells were harvested and relative mRNA levels were quantified by qRT-PCR. The

application of dCas9-VPR compared to dCas9-VP64 resulted in increased expression

of the target gene IL1RN while using CRISPRa (Fig. 4 A). For endogenous activation

of IL1RN, when compared to the dCas9-VP64 activator, dCas9-VPR activated the gene

by a ~1000-fold increase. Through this, we confirmed that the dCas9 system activates

endogenous gene targets and dCas9-VPR has higher induced gene activation than

dCas9-VP64. Schematically, four different sgRNAs recruit dCas9-VPR to the promoter

region of IL1RN as shown in Figure 4 B. The black boxes of the intron-exon map

indicate the coding exons while the white boxes indicate the non-coding exons.

Distances between sgRNAs and exon boxes indicate the sequence length between them.

3.3 Design of light-inducible CRISPRa system (LICAS)

based on CRY2FL-VPR and CIBN-dCas9-CIBN

In recent years, dynamic control of systems via light activation has grown in popularity

and been improved. For the first time, we constructed CRY2FL-VPR and CIBN-dCas9-

CIBN plasmids to optically control gene expression and cellular behavior in an

17

inducible and repeatable manner. For LICAS, the heterodimerization protein CRY2FL

and CIBN were fused to dCas9 and VPR to achieve endogenous gene upregulation

under the stimulus of blue light. The CRY2FL-VPR and CIBN-dCas9-CIBN plasmids

were cloned using Gibson Assembly.

VPR was digested with SacII and BstEII to remove the dCpf1 sequence and amplified

by PCR (Fig. 5). Additionally, it was assembled into a pcDNA3.1 vector containing

CRY2FL-VP64. The CRY2FL-VPR plasmid clone was digested with EcoRI and the

digested DNA was subjected to agarose gel electrophoresis to separate the cloned

fragments from the plasmid vector DNA (Fig. 6).

The CIBN-dCas9-CIBN plasmid was cloned by digesting pcDNA3.1-dCas9-VPR with

XbaI and AscI and using Gibson Assembly to insert the PCR-amplified CIBN gene and

create pcDNA3.1-dCas9-CIBN.

3.4 Activation of an exogenous eGFP reporter by CRY2FL-

VPR.

To validate the functionality of the CRY2FL-VPR plasmid in LICAS, we targeted an

exogenous eGFP reporter plasmid (Fig. 7 A) to obtain a visual and quantitative result.

We found that CRY2-VPR modestly activated gene expression of eGFP under blue light

illumination. However, contrary to our expectations, CRY2FL-VP64 produced higher

gene expression of eGFP and IL1RN than CRY2FL-VPR (Fig. 7 B). We believe this

discrepancy can be explained by the much larger molecular size of VPR when

compared to VP64.

18

3.5 Comparison of CRY2FL-VPR to CRY2FL-VP64 in

LICAS.

After confirming the functionality of the cloned CRY2FL-VPR constructs in the

exogenous gene expression system, its gene activation levels were tested in the

endogenous gene expression system by targeting IL1RN in HEK293T cells (Fig. 8 A).

To validate the functionality of CRY2FL-VPR, HEK293T cells were seeded at ~80%

confluency on a 12-well plate one day before transfection. After 24 hours of transfection,

cells were illuminated with blue light for 48 hours and then harvested and analyzed for

qRT-PCR. Our results indicated that when compared to CRY2FL-VPR, CRY2FL-VP64

induced IL1RN gene activation by an increase of ~22-fold. (Fig. 8 B). This result shows

that the stand-alone efficiency of an activator does not necessarily lead to an

enhancement of LICAS. Possible explanations are further explored in the discussion

section.

4. Discussion

Cells dynamically respond to cellular events and signals from their surroundings and

adapt according to cellular needs. Therefore, to control gene expression in a time-wise

and situational manner, researchers are using inducible gene activation systems to

upregulate gene expression and change the behavior of specific cells [28]. The recently

developed light inducible CRISPR activation system allows for time dependent and

endogenous expression of genes and is seen as a powerful, potential tool to advance

gene engineering.

Native gene regulation is highly dynamic, as transcription levels of genes can change

19

during cell differentiation and across cell types. Over the last several decades,

researchers have assembled inducible tools that enable control of gene expression

through the introduction of exogenous plasmids into cells [29]. This method has been

effective but notably, can have toxic adverse effects [30]. This factor renders

spatiotemporal control of genes through exogenous activation a non-ideal method for

humans. Gene regulation tools must be safe and simple to have clinical viability.

Furthermore, tools themselves should not interfere with non-targeted endogenous genes

or affect normal cellular signal systems.

Therefore, we chose to improve a light-inducible system. A light-inducible system is

relatively noninvasive and inducible [31]; the desired effect can be precisely controlled

through the use of a blue light source. Compared with other inducible methods, a light-

inducible method has shown to be more convenient and easier to manipulate [32]. Blue

light is used to control the interaction of light-inducible proteins; proteins that have been

previously exploited in genome engineering [10]. To construct LICAS, we first adapted

a light-inducible Cryptochrome 2 (CRY2FL) and its CIBN partner protein (both derived

from Arabidopsis thaliana) to bind to each other in response to 470nm blue light. When

blue light is absent, CRY2FL and CIBN proteins exist in a separated state, with the

CRISPRa system nonfunctional; when blue light is on, they form a heterodimer and the

activator domain (in our case VPR or VP64) successfully binds to the promoter region

to initiate gene activation.

We constructed CRY2FL-VPR through Gibson Assembly and examined VPR and

VP64’s working efficiencies in CRISPRa and LICAS. In CRISPRa, VPR is directly

fused to dCas9 and showed ~1000-fold higher activation of IL1RN than VP64. However,

in LICAS, after being fused to CRY2FL and forming CRY2FL-VPR, IL1RN activation

by VPR was lower than VP64 by a factor of ~22-fold. We affirm that our construction

20

of CRY2FL-VPR enabled light-inducible transcriptional activation of exogenous and

endogenous gene targets. However, compared to CRY2FL-VP64, CRY2FL-VPR’s

activation efficiency was modest for LICAS. We found that, in order to improve LICAS,

the stand-alone efficiency of VPR was not enough or did not amend the bottleneck of

the system. Our result demonstrated that although tripartite transcriptional activator

VPR is significantly more efficient than single domain transcriptional activator VP64

in the conventional CRISPRa system, this result was not reproduced in LICAS. We

believe external factors, such as transcriptional activator size and its interaction with the

fused heterodimers CRY2FIL and CIBN, adversely affected the LICAS’ efficiency. We

attend that this discrepancy can be explained by the much larger molecular size of VPR

when compared with VP64 [23]. Based on our observation and results, we surmise that

because the efficiency of a transcriptional activator is heavily influenced by its

molecular characteristics, such as molecular size, being fused to a functionally mobile

heterodimerizing protein CRY2FL negatively affected VPR’s ability to induce gene

activation [21, 24, 33, 34]. Due to CRY2FL-VPR and CRY2FL-VP64 having highly

mobile molecular interactions in LICAS, CRY2FL-VPR’s large size may have slowed

its binding interaction with CIBN-dCas9-CIBN. Compared with dCas9-VPR and

dCas9-VP64’s less mobile molecular interaction in CRISPRa, CRY2FL-VPR and

CRY2FL-VP64 must move and bind efficiently in LICAS. VPR is 7 times larger

(~1077bp or 38.5kDa) than VP64 (~150bp or 5,5kD). Therefore, this discrepancy

possibly inhibited CRY2FL-CIBN heterodimerization that is required for the

functioning of LICAS.

We believe that gene activation efficiency of LICAS may still be increased through 1)

the improvement of fusion design between CRY2FL and VPR and 2) better construction

of the blue light illumination system. Researchers should first consider investigating the

21

modification of the CRY2FL-CIBN heterodimers with their truncated versions.

Truncated versions of these proteins were previously found to promote robust light-

dependent interaction; generally reducing the length of DNA or decreasing the bulk size

of proteins can help them interact more efficiently [21]. Researchers modified the full-

length CRY2FL into a shorter version CRY2PHR (residues 1–498 of CRY2) and found

improved expression over the full-length CRY2 [21]. Therefore, we suggest

improvements to the overall LICAS system by using truncated versions of the

heterodimerizing proteins or reducing the size of the components (smaller

transcriptional activator) that promotes the CRISPR-dcas9 interaction.

In conclusion, highly precise regulation of gene activation using LICAS will enable

researchers to investigate the causal role of gene regulation in diverse processes

including cell development and disease treatment. This investigation can be heavily

used in applied biology and has potential to contribute to clinical gene therapy. We

demonstrate that the CRY2FL-VPR system establishes an alternative method of

optogenetic control for endogenous gene activation.

22

Figures and Tables

Figure 1. Major application areas of CRISPR-Cas9 based technologies beyond genome

editing. (AdliMazhar, 2018)

Figure 2. Schematic view of the programmable CRISPR-dCas9-based transcriptional

activator systems. (A) The tripartite transcriptional activator VPR in CRISPRa system.

23

(B) The tripartite transcriptional activator VPR in a light-inducible CRISPR activation

system (LICAS). After blue light illumination, heterodimers interact with each other

and transport VPR to the target gene’s promoter site and intiates transcription.

Figure 3. Construction of LICAS – blue light LED circuit establishment.

24

A

B

Figure 4. Comparison of dCas9-VP64 and dCas9-VPR in a CRISPRa system to

activate endogenous genes. (A) Table of nucleotide sequence for IL1RN and consequent

sgRNA design. (B) IL1RN. NT: no transfection. Data shown is combined triplicate

qRT-PCR results (error bars indicate the s.e.m.). Analysis done by two-tailed students

t-test, p-values of less than 0.05 were considered significant (ns = no significance, * =

p-value < 0.05, *** = p-value < 0.001).

25

Figure 5. Design and construction of a cloning vector CRY2FL-VPR through Gibson

Assembly.

26

Figure 6. A visualization of restriction fragments CRY2FL-VPR separated by gel

electrophoresis.

27

Figure 7. Exogenous eGFP expression in LICAS. (A) Construction design of

exogenous eGFP reporter with minimal CMV promoter and eight repeats of sgRNA

targeting upstream sites of eGFP (B) Cells processed by LICAS were either incubated in

the dark (Dark) or illuminated (Light) for 24 hours. Cells were transfected with the eGFP

reporter and an empty plasmid as a negative control. When the blue light is illuminated,

the eGFP gene was activated by the dimerization of CRY2FL-VP64 and CRY2FL-VPR

with CIBN-dCas9-CIBN.

28

A

B

Figure 8. Activation of IL1RN via LICAS. (A) Overall experimental flow of LICAS.

(B) Gene expression of LICAS and CRISPRa system with respect to VP64 and VPR

effector.

29

Table 1. Optimization result of electroporation

Electroporation parameters

Pulse

voltage

(V)

Pulse

with

(ms)

Pulse

number

Cell

density

(cells/ml)

Transfection

efficiency Viability Tip type

1100 2 2 1× 105 80% 80% 100 µl

Table 2. Sequence of the primers used for qRT-PCR.

Gene of

interest

Primers

Forward Reverse

IL1RN GGAATCCATGGAGGGAAGAT TGTTCTCGCTCAGGTCAGTG

30

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