Establishing a CRISPR screening platform for metabolic driver IncRNAs in lung cancer and setting up a glucose metabolism

assay for high-throughput screening Juliette Schlatter, BMA 16-19

College of Higher Education

Educational Program for Biomedical Scientists

Department for Biomedical Research (DBMR), Berne, Switzerland

1. Abstract

Being one of the most common cancers lung cancer is newly diagnosed in approximately 2’500 men and 1’200 women each year in Switzerland. 85% of all these

lung cancer cases account for NSCLC (Non-small cell lung cancer). NSCLC is often associated with a mutation that occurs in the gene KRAS, which comes along

with increased resistance in conventional chemotherapies. Specific treatments for NSCLC with KRAS mutation do not presently exist, which is why researchers

are trying to investigate IncRNAs (long non-coding RNA’s) as new therapeutic targets in NSCLC. Aim of this thesis is to develop Cas9 expressing H1299, H460

and A427 NSCLC cell lines as a platform for screens of such metabolic driver IncRNAs in lung cancer. Wild type cells were transfected with the Cas9 protein

sorted by fluorescence activated cell sorting and validated with qPCR. To determine an involvement of the found IncRNAs in cancer metabolism, a glucose

metabolism assay was established in the A549 cell line simultaneously as a possibility for phenotyping by cloning individual CRISPR targeting constructs and

using a fluorescent glucose derivate, the 2- NBDG. The platform could be successfully established and validated. The setup of the glucose metabolism assay

proved to be difficult but could be validated with the cloned constructs as well. For better evaluation however, more measurements need to be performed. Both,

the Platform of Cas9 expressing NSCLC cell lines and the glucose metabolism assay, now lay the foundation for future screens and experiments of the laboratory.

4. Material and Methods

3. Aims

The development of a glucose metabolism assay may be used as a method to

verify if IncRNAs can affect the metabolism of cancer cells. A new compound,

the 2 NBDG (fluorescent 2-deoxyglucose analogue 2- [N-(7-nitrobenz-2- oxa-

1,3-diazol-4- yl) amino]-2-deoxyglucose) will be used to visibly validate any

changes in the glucose metabolism. Cells (A549) that are used for this

experiment need to express the Cas9 protein!

Leading Question

- Can I establish a glucose metabolism assay using the 2-NBDG and the

Cas9 expressing A549 cell line, that can be deployed as screening method

for metabolic changes in cancer cells?

Visualized workflow of all steps and methods of the project:

2. IntroductionWarburg Effect

To promote proliferation, survival, long-term maintenance and cell growth

cancer cells alter their metabolism. The first alteration and biochemical

hallmark of tumour cells that has been described is the increased glucose

uptake and a shift in their method of producing energy in form of ATP [1]. The

effect of switching from the mitochondrial oxidative phosphorylation to

fermentation of glucose to lactate, even in the presence of oxygen and fully

functioning mitochondria, is known as the ‘Warburg Effect’ and a common

feature of cancer cells [2]. Cancer cells therefore alter the glucose metabolism

by affecting metabolic enzymes or kinases. Genes involved in the glucose

uptake and metabolism could therefore be promising therapeutic targets to

decelerate the proliferation and hamper the survival of cancer cells [3].

CRISPR/Cas9 Knock-Out

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the

non-specific CRISPR-associated endonuclease protein Cas9 were used as an

efficient tool for gene editing. Cas9 can recognise and cut any target DNA with

a high degree of specificity by using a short RNA fragment, also called guide

RNA (gRNA). The endonuclease creates a double-strand break at this target

region, which results in mutations that permanently disrupts a gene so no

functional protein can be produced anymore [4]. For this project individual

CRISPR targeting constructs for genes involved in glucose metabolism were

cloned and introduced in Cas9 expressing A549 cancer cells.

5. R

First priority of this thesis was to establish a functioning glucose metabolism

assay. The chosen genes thus only work as verification for the working assay.

The control used (#1806) describes a targeting construct, which is a

CRISPR/Cas9 Plasmid. It targets a location of the cells genome that is known

of not changing their metabolism in any way. Each gene was cloned into two

constructs except Glut1 and PTEN, where only one gRNA worked for the

experiment.

References: [1] Shaw, R. J. (2006, December 06). Glucose metabolism and cancer. Retrieved from Current

Opinion in Cell Biology: https://doi.org/10.1016/j.ceb.2006.10.005

[2] Vander Heiden, M. G., Cantley, L. C., & Thompson, C. B. (2009, May 22). Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation. Retrieved from Science:

https://www.doi.org/10.1126/science.1160809

[3] Fan, C., Tang, Y., Wang, J., Xiong, F., Guo, C., Wang, . . . Zeng, Z. (2017, July 11). Role of long non-coding RNAs in glucose metabolism in cancer. Retrieved from Molecular Cancer:

https://www.doi.org/10.1186/s12943-017-0699-3

[4] Doudna, J. A., & Charpentier, E. (2014, November 28). The new frontier of genome engineering with CRISPR-Cas9. Retrieved from Science: https://www.doi.org/10.1126/science.1258096

[5] Esposito, R., Bosch, N., Andrés, L., Polidori, T., Puido-Quetglas, C., & Johnson, R. (2019, April

15). Hacking the Cancer Genome: Profiling Therapeutically Actionable Long Non-coding RNAs Using CRISPR-Cas9 Screening. Retrieved from https://doi.org/10.1016/j.ccell.2019.01.019

Define glucose metabolism

related genes as controls for

the assay

Design gRNA for each gene

using CRISPETa [5] and cloning

into pDECKO-mCherry Plasmid

Transfection of Plasmid into

Cas9 expressing A549 cells

Selection with Puromycin

(antibiotic) and checking the

knock out with genomic DNA

extraction and following PCR

FACS (Fluorescent-activated cell

sorting) using established 2-NBDG

glucose Metabolism assay

Establish the glucose metabolism

assay using 2-NBDG (a

fluorescence marked glucose

derivate)

1. Abstract

2. Introduction 5. Results

3. Aims

4. Material and Methods

0

20

40

60

80

100

120

#1806 Glut1

(SLC2A1)

HK n1 HK n2 AKT n1 AKT n2

Upta

ke o

f 2

-NB

DG

in

% in

com

pariso

n t

o #

1806

Glucose uptake increasing genes compared to #1806*

0

20

40

60

80

100

120

140

#1806 PLIN2 n1 PLIN2 n2 LINC00473 n1LINC00473 n2 PTEN

Upta

ke o

f 2-N

BD

G in

% in

com

parison to #

1801

Glucose uptake reducing genes compared to #1806

Table 5.1: Shown are the genes that increase the glucose uptake in cancer cells. In our CRISPR KO cells this

genes have been silenced and a decrease in glucose uptake was expected. Glut1 KO cells have been

observed to change their uptake significantly although in the opposite direction as was expected.

Other genes behaved as expected, although not significantly.

Table 5.2:Shown are the genes that decrease the glucose uptake in cancer cells. In our CRISPR KO cells this

genes have been silenced and an increase in glucose uptake was expected. Although higher uptake

of 2-NBDG uptake was predicted there is little to no change visible for the investigated genes.

N=2

P-value = 0,05

N=2

P-value = 0,05

Defining phenotypes of genes is regularly done in laboratories using

proliferation -and wound healing assays. With the newly established glucose

metabolism protocol the laboratory has gained another option of screening for

metabolic changes in cancer cells caused by the IncRNAs, which are being

investigated as new therapeutic targets in NSCLC.

Establishing the assay proved to be time consuming and difficult since the 2-

NBDG has not yet been applied using FACS for visible validation. Hence,

after completing the finally working protocol, only little time remained for

measurements with the individual cloned CRISR constructs (n=2). To validate

the data in a better way more measurements would need to be performed

and the unexpected results concerning the GLUT1 should be investigated.

6. Discussion

Figures:

DNA Figure:

http://miror-ejd.eu/news/

Tables:

Table 5.1 Glucose uptake increasing

genes compared to #1806, own table

Table 5.2 Glucose uptake reducing

genes compared to #1806, own table