APOL1 the most common causes of primary

APOL1 variants are very frequent and have substantial
penetrance, unlike other complex disease genes. The strongest effect size
recorded for APOL1 variant associations with kidney disease is for HIVAN (HIV
associated nephropathy). Studies have shown that the strong effect sizes (up to
29) and the high frequency (up to 72%) of cases that carry two risk alleles for
HIVAN, primary focal segmental glomerulosclerosis (FSGS), Lupus collapsing
glomerulopathy, sickle cell disease nephropathy, and hypertension-attributed
arterionephrosclerosis. The three main goals are to figure out how APOL1 alter
kidney physiology, why APOL1 gives immunity to trypanosomal illness, and how to
genetically modify the APOL1 allele in patients in order to prevent the
development of FSGS. (Limou)

 

Background:

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The first cases of (FSGS) were found in 1957. It is one of
the most common causes of primary glomeruli in older people. “Over the years,
it has increased and caused asymptomatic proteinuria or nephrotic syndrome with
or without renal insufficiency”(Rao). By going after the glomeruli in your
kidney, the small units that clean the blood, FSGS causes scarring in your
kidney. This scarring affects males more than it affects women and happens
frequently in African Americans. There are two types of FSGS: primary FSGS,
which happened on its own, and secondary FSGS which was caused by another
disease or a drug.  Treatment for FSGS is
using Corticosteroids, also known as steroids, immunosuppressive drugs, diet
change and more (Rao).

CRISPR is a bacterial immune system that knocks out a DNA
strip and replaces it with a “donor” DNA strip. Cas 9 allows it to go into the
cell and manipulate the DNA strand. Guide RNA has the same gene sequence as the
strip wanting to be replaced and Cas 9 makes it happen. APOL1 is the gene we
are trying to replace. APOL1 is a somewhat large plasma protein and is the more
significant apoprotein of HDL. Causing the flowing out of cholesterol from
cells and being involved in the creation of the majority of cholesteryl esters,
APOL1 also exchanges lipids throughout the body and reverses the cholesterol
movement from outer cells to the liver (Matsha).

 

Plan:

Recognizing that using CRISPR-CAS9 to “knock out” the APOL1
variant and replace the strip of DNA with the APOL1 with a donor strip, it will
allow the DNA to be modified and be cured of Focal Segmental
Glomerulosclerosis. 

A deeper understanding is gained by conducting research and
experiments of the background of the APOL1 gene and how CRISPR is used to alter
genes and how it can be used to cure FSGS disease. Such experiments are as
follows:

1.   
Lipofectamine 2000 DNA transfection Reagent Protocol: Seed cells into
96-well plate.  Dilute four tubes of
Lipofectamine Reagent with the Opti-MEM medium. In a separate tube, dilute
DNA—from bacteria plasmid—with the Opti-MEM medium. Add diluted DNA and diluted
Lipofectamine Reagent together. After, incubate for 5 minutes in the incubator.
Add the stock of the DNA and Lipofectamine to cells. Incubate for 1-3 days.
Dilute FTY720 with the drug concentrations of its Ic50. Add FTY720 dilutions to
the cell. After analyzing transfected cells with operetta.

2.   
Western Blot: Treat cells Doxorubicin. Treat cells with the IC50
concentration of FTY720. Incubate for 24 hours. Prepare whole cell lysates.  Run SDS-polyacrylamide gel electrophoresis to
separate proteins based on size. Transfer separated proteins on polyacrylamide
gel onto a membrane.  Block membrane
using Blotto.  Incubate membrane with one
of the following primary antibodies. Rinse membrane to remove unbound
antibodies. Incubate membrane with secondary antibody conjugated with a
fluorescent probe. Rinse membrane to remove unbound antibodies. Proteins were
detected using the Odyssey Infrared Imaging System (LI-COR Biosciences,
Lincoln, NB, USA) and analyzed with Licor Image Studio 2.0 acquisition and
analysis software.

 

Solution/Cure:

–       the solution in curing FSGS is using
CRISPR-CAS9.

–       The portion of DNA with the APOL1
variant that is partially responsible for the development of FSGS would be replaced
using CRISPR-CAS9.

To cure Focal segmental glomerulosclerosis (FSGS), doctors
and scientist must consider using CRISPR-CAS9. CRISPR-CAS9 is a bacterial
immune system that is able to “knock off” an undesirable gene, changing the
genetic makeup. CRISPR allows a foreign DNA to insert itself into a cell and
able to identify the undesirable gene with CAS 9. Once it identifies the
unwanted genes, it will “knock off” the gene and replace it with a “donor”
gene. One way scientist is able to perform this experiment is using
transfection and Western Blot. Using Lipofectamine is a transfection reagent
and mixing it with the CRISPR-CAS9, the lipofectamine will allow the
CRISPR-CAS9 to insert itself into foreign cells. Once the CRISPR-CAS9 is inside
the cell, the CRISPR can go to work and “knock off” the unwanted gene. To
ensure the certain gene is no longer there, conducting a Western Blot
experiment is expected. Western Blot requires SDS-polyacrylamide gel
electrophoresis, which will be used to identify that the protein will no longer
is expressed. Inside of the gel electrophoresis are proteins that are mixed
with primary and secondary antibodies; these proteins are then used to identify
the unwanted protein. SDS is then placed in the Odyssey Infrared Imaging
System, where it reads the proteins. Once it is done scanning you’re able to
identify the concentration the gene is expressing. A low amount of
concentration means there is a smaller amount of the gene within the DNA
structure. (Lange) This is our group’s solution and experiment in curing FSGS.

 

Summary:

The APOL1 gene was discovered and has improved the
understanding as to what mainly causes kidney disease. Our solution to “fixing”
the problem with the APOL1 variant was to knock off the APOL1 gene using
CRISPR-CAS9. By knocking off the gene with CRISPR-CAS9, the genetic makeup of
the DNA is changed. Thus, giving the cells a chance it defeating 

APOL1 variants are very frequent and have substantial
penetrance, unlike other complex disease genes. The strongest effect size
recorded for APOL1 variant associations with kidney disease is for HIVAN (HIV
associated nephropathy). Studies have shown that the strong effect sizes (up to
29) and the high frequency (up to 72%) of cases that carry two risk alleles for
HIVAN, primary focal segmental glomerulosclerosis (FSGS), Lupus collapsing
glomerulopathy, sickle cell disease nephropathy, and hypertension-attributed
arterionephrosclerosis. The three main goals are to figure out how APOL1 alter
kidney physiology, why APOL1 gives immunity to trypanosomal illness, and how to
genetically modify the APOL1 allele in patients in order to prevent the
development of FSGS. (Limou)

 

Background:

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The first cases of (FSGS) were found in 1957. It is one of
the most common causes of primary glomeruli in older people. “Over the years,
it has increased and caused asymptomatic proteinuria or nephrotic syndrome with
or without renal insufficiency”(Rao). By going after the glomeruli in your
kidney, the small units that clean the blood, FSGS causes scarring in your
kidney. This scarring affects males more than it affects women and happens
frequently in African Americans. There are two types of FSGS: primary FSGS,
which happened on its own, and secondary FSGS which was caused by another
disease or a drug.  Treatment for FSGS is
using Corticosteroids, also known as steroids, immunosuppressive drugs, diet
change and more (Rao).

CRISPR is a bacterial immune system that knocks out a DNA
strip and replaces it with a “donor” DNA strip. Cas 9 allows it to go into the
cell and manipulate the DNA strand. Guide RNA has the same gene sequence as the
strip wanting to be replaced and Cas 9 makes it happen. APOL1 is the gene we
are trying to replace. APOL1 is a somewhat large plasma protein and is the more
significant apoprotein of HDL. Causing the flowing out of cholesterol from
cells and being involved in the creation of the majority of cholesteryl esters,
APOL1 also exchanges lipids throughout the body and reverses the cholesterol
movement from outer cells to the liver (Matsha).

 

Plan:

Recognizing that using CRISPR-CAS9 to “knock out” the APOL1
variant and replace the strip of DNA with the APOL1 with a donor strip, it will
allow the DNA to be modified and be cured of Focal Segmental
Glomerulosclerosis. 

A deeper understanding is gained by conducting research and
experiments of the background of the APOL1 gene and how CRISPR is used to alter
genes and how it can be used to cure FSGS disease. Such experiments are as
follows:

1.   
Lipofectamine 2000 DNA transfection Reagent Protocol: Seed cells into
96-well plate.  Dilute four tubes of
Lipofectamine Reagent with the Opti-MEM medium. In a separate tube, dilute
DNA—from bacteria plasmid—with the Opti-MEM medium. Add diluted DNA and diluted
Lipofectamine Reagent together. After, incubate for 5 minutes in the incubator.
Add the stock of the DNA and Lipofectamine to cells. Incubate for 1-3 days.
Dilute FTY720 with the drug concentrations of its Ic50. Add FTY720 dilutions to
the cell. After analyzing transfected cells with operetta.

2.   
Western Blot: Treat cells Doxorubicin. Treat cells with the IC50
concentration of FTY720. Incubate for 24 hours. Prepare whole cell lysates.  Run SDS-polyacrylamide gel electrophoresis to
separate proteins based on size. Transfer separated proteins on polyacrylamide
gel onto a membrane.  Block membrane
using Blotto.  Incubate membrane with one
of the following primary antibodies. Rinse membrane to remove unbound
antibodies. Incubate membrane with secondary antibody conjugated with a
fluorescent probe. Rinse membrane to remove unbound antibodies. Proteins were
detected using the Odyssey Infrared Imaging System (LI-COR Biosciences,
Lincoln, NB, USA) and analyzed with Licor Image Studio 2.0 acquisition and
analysis software.

 

Solution/Cure:

–       the solution in curing FSGS is using
CRISPR-CAS9.

–       The portion of DNA with the APOL1
variant that is partially responsible for the development of FSGS would be replaced
using CRISPR-CAS9.

To cure Focal segmental glomerulosclerosis (FSGS), doctors
and scientist must consider using CRISPR-CAS9. CRISPR-CAS9 is a bacterial
immune system that is able to “knock off” an undesirable gene, changing the
genetic makeup. CRISPR allows a foreign DNA to insert itself into a cell and
able to identify the undesirable gene with CAS 9. Once it identifies the
unwanted genes, it will “knock off” the gene and replace it with a “donor”
gene. One way scientist is able to perform this experiment is using
transfection and Western Blot. Using Lipofectamine is a transfection reagent
and mixing it with the CRISPR-CAS9, the lipofectamine will allow the
CRISPR-CAS9 to insert itself into foreign cells. Once the CRISPR-CAS9 is inside
the cell, the CRISPR can go to work and “knock off” the unwanted gene. To
ensure the certain gene is no longer there, conducting a Western Blot
experiment is expected. Western Blot requires SDS-polyacrylamide gel
electrophoresis, which will be used to identify that the protein will no longer
is expressed. Inside of the gel electrophoresis are proteins that are mixed
with primary and secondary antibodies; these proteins are then used to identify
the unwanted protein. SDS is then placed in the Odyssey Infrared Imaging
System, where it reads the proteins. Once it is done scanning you’re able to
identify the concentration the gene is expressing. A low amount of
concentration means there is a smaller amount of the gene within the DNA
structure. (Lange) This is our group’s solution and experiment in curing FSGS.

 

Summary:

The APOL1 gene was discovered and has improved the
understanding as to what mainly causes kidney disease. Our solution to “fixing”
the problem with the APOL1 variant was to knock off the APOL1 gene using
CRISPR-CAS9. By knocking off the gene with CRISPR-CAS9, the genetic makeup of
the DNA is changed. Thus, giving the cells a chance it defeating 

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