In Vivo Gene Modification by CRISPR/Cas-9 System

Claudia Arenas1, Chang Liu2, Dr. Qi-Long Ying3

1Bravo Medical Magnet High School & USC CIRM STAR Program, 2 Department of Stem Cell Biology and Regenerative Medicine, 3Keck School of Medicine of Medicine; University of

Southern California, Los Angeles, CA USA

Cloning the T-vector makes it possible to begin mutagenesis. Sequencing results demonstrated that our cells have the sequence of what Figure 5.2 will be. Mutagenesis has been started using the sequenced results. The sequence that we currently have will be used as a template to generate Figures 5(a) and 5(d). The PNPLA3 sequence for Figure 5(c) will be generated from Figure 5(a). Once the four combinations are generated, HepG2 cells will be transfected to create 4 different cell plates under the same conditions - in vitro NAFLD – as shown in Figure 5. Calculating exact fat values is still being considered so results from the cell cultures will first be only qualitative.

Background

Objective

Methods

Results

Discussion

Future Direction

Acknowledgements

Non Alcoholic Fatty Liver Disease (NAFLD) is when “excess fat accumulates in the liver of a patient without the history of alcohol abuse.” Although exact causes are unknown, obesity, rapid weight loss, insulin resistance (diabetes), and genetic factors are correlated to the disease. Once a person develops NAFLD, if left untreated, it can progress into Steatosis, Steatohepatitis, Fibrosis, Cirrhosis, and even death.

Determine the effect of different combinations of these SNPS on an ethnic group not known

to be affected by these SNPs

Figure 1 shows the sequencing results of the genomic DNA of HepG2 cells. Figure 2 is the result of mRNA from HepG2 cells. In both the genomic DNA and mRNA results, Figures 1(a) and 2(a), Methionine was present at the 148th codon instead of Isoleucine. SNP 1 exists in the HepG2 cells. Figures 1(b) and 2(b), genomic DNA and mRNA respectively, had the same results, SNP2 was not present at the 453rd codon. Theoretically, having SNP 1 but not SNP2 should make a person much more susceptible to NAFLD.

Dr. Qi-Long Ying, PIChang Liu, MentorYing Lab MembersDr. Roberta Diaz Brinton, Program DirectorChristina Zeitountsyan, Program CoordinatorMrs. Ramirez – De La Cruz, Program TeacherCIRM and STAR Program

The patatin like phospholipase domain containing 3 (PNPLA3) gene encodes a lipase that mediates hydrolysis in adipocytes. If the protein is not translated, the breakdown of fat is disrupted in the liver, resulting in fat accumulation and the liver damage without any alcohol abuse.In 2009, PNPLA3 gene was identified as significant to certain ethnic groups’ susceptibility to NAFLD. The I148M variant, a single nucleotide polymorphism (SNP), was found to increase Hispanics‘ susceptibility to the disease. The second SNP, the S453I variant, was found to make African Americans less susceptible to the disease. The HepG2 cells that will be used in this project are an immortalized cell line that come from a young, Caucasian male. Because it is unknown what the effect of the variants are in other ethnic groups, this is significant.

Hypothesis

Fig. 1 Sequencing results of genomic DNA from HepG2 cells (a) SNP is present at the 148th codon resulting in change from Isoleucine to Methionine.

The red square indicates a mismatch with the sequence of the program.(b) SNP not present at the 453rd codon so Serine remains the same

Isoleucine&

Serine

No SNP (Wild Type)

Methionine&

Isoleucine

SNP 1&2

Isoleucine&

Isoleucine

SNP 2

Methionine&

Serine

SNP 1

Of the four combinations of SNP’s the one with SNP 1 but without SNP 2 should retain the

most fat.

Figures 5 (a)-(d) demonstrate the different combinations of SNPs.

(a) (b) (c) (d)

(b)(a)

Fig. 2 Sequencing results of mRNA from HepG2 cells (a) SNP is present at the 148th codon resulting in change from Isoleucine to Methionine.(b) SNP not present at the 453rd codon so Serine remains the same

(a) (b)

1. Extract genomic DNA from HepG2 cells and determine sequence at 148th and 453rd codon

2. Extract mRNA from HepG2 cells and generate variants of PNPLA3

HepG2 cells

Extract genomic

DNA Send for

sequencing

Extract mRNA

PCR to get cDNA from

mRNA

T-vector cloning

vector

insert

Mutagenesis