Unlocking Hope: CRISPR’s Promise for Cystic Fibrosis Treatment

Julia Fitlin, Kristen Bradish, Heather Horowitz

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Proposal Keywords:
1. Cystic Fibrosis
2. CRISPR/cas9
3. Gene editing
4. CFTR gene
5. Homology-directed gene repair
Summary:

Individuals with Cystic Fibrosis (CF) face significant health challenges, and shortened life

spans due to respiratory and digestive complications1. Our study is needed because it

addresses a critical medical need by offering a potential cure for this devastating disease. This

genetic recessive disorder is most commonly caused by a deletion of the F508 amino acid on

the CFTR gene which produces a full-length but misfolded gene that gets degraded in the

endoplasmic reticulum. Despite advancements in gene therapy, there are still challenges in

delivering therapeutic genes to the target epithelial lung cells efficiently and safely2. Our project

aims to use a CRISPR/cas9 system for the targeted insertion of a full-length CFTR cDNA into

the CFTR locus to replace the endogenous CFTR through directed homologous gene repair. To

do this, we will first obtain CRISPR/cas9 DNA, CFTR template strand, GFP, and sgRNA

biobricks. Then, we will insert the CFTR template strand and GFP into a biobrick plasmid that

has chloramphenicol resistance and the sgRNA and cas9 into a separate biobrick plasmid also

treated with antibiotic resistance. Then, Sanger Sequencing will verify if the genes were

successfully implemented into the plasmids. The plasmids will then be transfected into epithelial

cells, grown on a chloramphenicol-treated plate, detected for fluorescence, and lysed to extract

the DNA. If fluorescence is detected, the cells will glow when exposed to UV light. Then, we will

perform PCR genotyping on our plasmids to confirm the knockin gene is present. We will finally

run gel electrophoresis and see if there is a strong band presence where our gene should be.

Following this, we will use the modified solvent evaporation method to get both plasmids into the

lipid nanoparticles (LNPs) to then suspend in solution, freeze dry, and lyophilize3. We will end by

taking our lyophilized solution, which now has fragile or easily breakable LNPs, suspend them in

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an aerosol solution, and deliver them through a pulmonary inhaler. The CRISPR/cas9 plasmid

and the CFTR plasmid in solution will travel through the pulmonary system to be delivered to

epithelial cells, specifically the HSAEC1-KT cells from the hTERT-immortalized cell line before

moving to living organisms. Our initial studies will include mice until we can validate the

treatment's safety and efficacy for humans. Our project will have significant benefits for patients

suffering from CF if our design works as planned. Successful completion of our study will

significantly advance our understanding of CRISPR/Cas9 technology and its application in

treating genetic diseases like CF. This approach could pave the way for developing similar

gene-editing therapies for other genetic disorders, thus advancing the field of genetic medicine.

Each patient's genetic mutation can be specifically targeted and corrected, allowing for tailored

therapeutic interventions that address the underlying cause of the disease, therefore bettering

the medical solution for CF. If successful, rather than relying solely on symptomatic

management, patients could experience long-term relief from respiratory and digestive

complications, leading to improved overall health outcomes and quality of life.

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Figure 1. Graphical Abstract The general process of our proposed plan and a depiction of how

this gene therapy would be delivered to humans.

Proposal Background:

The debilitating autosomal recessive genetic disease known as Cystic Fibrosis (CF) is

most distinguished by its severe effects on the respiratory system. It causes thick, sticky mucus

to accumulate in the airways, which worsens lung damage over time and results in recurring

infections and chronic inflammation. The CFTR gene, which codes for a protein essential for

controlling chlorine ion transport across cell membranes in many tissues, including the lungs, is

mutated in CF patients4. The malfunctioning CFTR protein is brought on by mutations in the

cystic fibrosis transmembrane conductance regulator (CFTR) genes in individuals with CF. The

protein cannot assist in transferring chloride- the primary component in salt- to the cell surface

when it is malfunctioning. Without chloride, which draws water to the cell surface, mucus in

different organs gets thick and sticky. Over two thousand gene variants related to the conditions

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have been found, with the F509del mutation being the most common4. The mucus clogs the

airways in the lungs and holds onto bacteria and other germs, which can cause infections,

inflammation, respiratory failure, and other problems. Because of this, staying away from

pathogens is very important to CF patients.

A 2022 analysis estimates that 162,428 people with CF reside in 94 countries5. The

illness, which is more common among Caucasian people, affects approximately 30,000 people

in the United States5. Although non-Hispanic white people are disproportionately affected by CF,

the condition is becoming more common among racial and ethnic minorities. Minority groups

with CF frequently have worse health outcomes than non-Hispanic whites, including lower lung

function and more excellent mortality rates, despite having equal socioeconomic status6. These

discrepancies are caused by several factors, including racial biases in healthcare,

underrepresentation in clinical trials, delayed diagnoses, and, most importantly, restricted

access to effective treatments.

Clinical investigations investigating aerosolized viral vectors, like AAV serotype 2

(AAV2), to deliver CFTR cDNA to the human lungs and nasal cavities in the early 2000s

revealed no clinically meaningful effect regarding lung function restoration7. However, the trials

were safe. Challenges included low transduction effectiveness in airway epithelial cells and

restricted packaging capacity of AAV27. Additionally, AAV’s strong immunogenicity presented

difficulties and prevented AAV-based treatments from being administered again because of

preexisting or developed immunity. Although AAV-based gene treatments showed promise for

treating CF, their success depends on overcoming immunogenicity and temporary expression

concerns.

Precision medicine could also be possible if gene editing technologies could correct

CFTR gene mutations and return them to normal functions. Early research on human intestinal

stem cell organoids and induced pluripotent stem cells produced from CF patients has shown

that CFTR function can be successfully restored. Recent developments included using an

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AAV-delivered CRISPR/cas9 platform to fix CFTR mutations in patient-derived airway basal

cells, resulting in nearly normal levels of CFTR activity7. However, despite these developments,

difficulties still exist because gene editing techniques are specialized to particular mutations,

necessitating customized repair for each mutation. However, these studies did show that

CRISPR can successfully repair a variety of CFTR abnormalities like slicing mutations and

premature termination codons, and it also holds promise for the advancement of cell-based

therapeutics for the disease7.

In recent years, there have been notable developments in CFTR-modifying therapy. With

the advent of ivacaftor, lumacaftor, and tezacaftor, approximately 50% more CF patients are

now eligible for CFTR-modifying medication ⁸. Regardless of their second mutation, CF patients

12 and older with at least one F508del mutation have hope. To improve CFTR protein function,

ivacaftor, a potentiator is combined with tezacaftor and elexacaftor, both correctors. However, a

major obstacle to access is its cost of approximately $24,000 each month8. One of our top

priorities is to create a more affordable and inclusive treatment for patients with cystic fibrosis.

Although current medications have improved the management of CF, certain patients

may not benefit from them due to specific mutation classes that are not impacted by current

therapies, tolerance concerns, expense, and continuous disease progression. To overcome

these restrictions and help CF patients return to normalcy, researchers need to look into

additional therapies and gene editing techniques like CRISPR-Cas technology.

Even though CF is currently incurable and has a substantial negative influence on life

expectancy, improvements in therapy have caused the average survival age to rise dramatically

to more than 40 years old9. As a result, CF is now acknowledged to impact both children and

adults and is no longer only thought to be a childhood illness. Since active therapy has been

demonstrated to improve quality of life, prolong life span, and improve prognosis, over half of

CF patients are now adults. In order to maximize the quality of life and long-term survival for CF

patients, there needs to be a push for new and effective technologies9.

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 CF is a severely life-hindering condition with persistent differences in treatment

outcomes. Advancements in gene editing technology and CFTR-modifying therapies hold

potential for better treatment outcomes and precision medicine methodologies. However,

guaranteeing fair access to care for all CF patients will require addressing problems like

excessive treatment costs and healthcare inequalities. For the field to advance and for the lives

of those who live with cystic fibrosis to improve, more research and innovation are essential.

Proposal Objective & Rationale:

We are proposing to create a gene therapy for patients living with Cystic Fibrosis, a

recessive genetic disorder that damages a person's lungs, pancreas, and other organs. Our

proposed project aims to use a CRISPR/Cas9 system for targeted insertion of a full-length

CFTR cDNA into the CFTR locus to replace the endogenous CFTR. This will be done through

directed homologous gene repair. The faulty CFTR gene is caused by deleting the F508 amino

acid on the gene that produces a full-length but misfolded CFTR gene degrading in the

endoplasmic reticulum. Our study is important because there are close to 40,000 children and

adults living with CF in the United States, and CF can affect people of every racial and ethnic

group¹⁰, so we need to find a solution for these people. Our study is also needed because it

addresses a critical medical need by offering a potential cure for this devastating disease.

Despite advancements in gene therapy, there are still challenges in delivering therapeutic genes

to the target cells efficiently and safely⁸. Our study is also needed because it addresses a critical

barrier to progress in gene therapy for CF and other genetic disorders. This project addresses a

significant problem in the field of CF treatment and offers a potential solution that could

revolutionize how this disease is managed and treated. Our project will be extremely impactful if

it works as expected. Successful completion of our study will significantly advance our

understanding of CRISPR/Cas9 technology and its application in treating genetic diseases like

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CF. Also, If successful, this approach could pave the way for developing similar gene-editing

therapies for other genetic disorders, thus advancing the field of genetic medicine. Each

patient's genetic mutation can be specifically targeted and corrected, allowing for tailored

therapeutic interventions that address the underlying cause of the disease, therefore bettering

the medical solution for CF. If this project works, rather than relying solely on symptomatic

management, patients could experience long-term relief from respiratory and digestive

complications, leading to improved overall health outcomes and quality of life.

Proposal Approach:

Our project aims to utilize CRISPR/Cas9 technology for targeted gene editing to address

cystic fibrosis (CF). We plan to insert a corrected CFTR gene into affected cells, replacing the

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faulty gene responsible for the disease. Initially, we will design and construct the necessary

genetic components, including the CRISPR/Cas9 system and the corrected CFTR gene. After

using Gibson Assembly and verifying their successful plasmid implementation, we will introduce

these constructs into target cells and confirm their uptake and expression. Following validation,

we will encapsulate these genetic constructs into nanoparticles for delivery. These

nanoparticles, designed to be easily administered via aerosol inhalation, will carry the corrective

genetic material to the affected cells.

Our proposed project aims to use a CRISPR/cas9 system for targeted insertion of a

full-length CFTR cDNA into the CFTR locus to replace the endogenous CFTR. This will be done

through directed homologous gene repair. This targeted gene repair approach addresses a

deletion mutation of the F508 amino acid, producing a misfolded CFTR protein prone to

degradation in the endoplasmic reticulum. CRISPR/Cas9 DNA, CFTR template strand, GFP,

and sgRNA biobricks will be obtained initially. The CFTR template strand and GFP will be

inserted into a biobrick plasmid with chloramphenicol resistance. In contrast, the CRISPR/Cas9

will be inserted into a separate plasmid with the same resistance marker. One biological part we

will be using from the registry of standard biological parts is BBa_K1218011, which is the cas9

enzyme needed to cleave the mutated gene. Another biological part that will be used from the

registry of standard biological parts is BBa_k2061002, which is the gRNA needed to bring the

enzyme and corrected gene to the point of mutation in the CFTR gene.

To make all the parts needed for our project work as a system after including them on

the plasmids mentioned above, we will perform the following steps to ensure successful

implementation. Sanger Sequencing will confirm successful gene implementation. The plasmids

will then be transfected into epithelial cells, grown on a chloramphenicol-treated plate, detected

for fluorescence, and lysed to extract DNA. If fluorescence is detected, the cells will glow when

exposed to UV light. Then, we will perform PCR genotyping on plasmids to confirm the knockin

gene is present. We will finally run gel electrophoresis and see if there is a strong band where

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our gene should appear. Subsequently, both plasmids will be incorporated into nanoparticles

using the modified solvent evaporation method. We will then suspend them in a solution that

freezes them dry and lyophilized them. Then we will take the brittle matrix, the now fragile or

easily breakable nanoparticles, and suspend them in aerosol solution and deliver it through a

pulmonary inhaler. The nanoparticles' CRISPR/Cas9 and CFTR plasmids will target lung

epithelial cells, offering a potential therapeutic strategy. Mouse models will be utilized for in vivo

testing.

Figure 2 Precise Design. This figure shows the creation of our CRISPR/cas9 plasmid made of

sgRNA, cas9, CFTR Gene, and Chloramphenicol Antibiotic Resistance. The plasmid will then

be injected into nanoparticles which will give us our PEGylated CRISPR/cas9 lipid nanoparticles

(LNPs).

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 Figure 3 Flowchart depicting our proposed approach in step-by-step detail.

Some expected outcomes are that we should see a strong band on the gel representing

our gene of interest for the PCR genotyping. The band size should be similar to that of the

control for a CFTR gene. Also, in cell culture, we would see the cells with our plasmid

fluorescence due to the expression of GFP. We should see that the CRISPR system works

inside cells and can cut out the mutated CFTR gene and replace it with a functioning copy. We

do this by looking at the protein expression from a western blot and DNA sequence from Sanger

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Sequencing. For Sanger Sequencing, we would see the corrected sequence if it worked and for

the Western blot, we would see a larger band representing the full CFTR protein.

We will make specific measurements throughout the experiment to ensure everything is

debugged correctly and works as designed. We will start by verifying that our recombinant

plasmids are successful through Sanger Sequencing. Our PCR genotyping and gel

electrophoresis experiment will confirm if our plasmid was transfected into the epithelial cells.

HPLC can be used to quantify the amount of plasmid encapsulated within each nanoparticle.

Finally, to ensure the CRISPR/cas9 system successfully cut and added the new gene, western

blot can confirm protein expression from the edited gene, and Sanger Sequencing can be used

to sequence the PCR products generated from the target region. If a new gene has been

successfully inserted, Sanger Sequencing will confirm its presence and sequence. We will also

have a negative control while optimizing our transfection conditions by removing the Cas

nuclease and only delivering the gRNA. This would mean the component that cuts DNA is not

present and therefore, no CRISPR edits will occur.

Literature Examples:

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Figure 4:

The figure depicts the validation of CRISPR/Cas9-mediated gene correction in primary cultured

intestinal stem cells derived from cystic fibrosis (CF) patients. Using forskolin-induced swelling

assays, the researchers confirmed the loss of CFTR function in CF patient-derived organoids.

Subsequently, CRISPR/Cas9 was employed to correct the CFTR mutation, resulting in restored

CFTR function, as evidenced by significant expansion of organoid surface area upon forskolin

treatment. This restoration of CFTR function was further validated through sensitivity to CFTR

inhibition, confirming the functional rescue of the CFTR phenotype in the corrected organoids. In

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this figure, F508del represents uncorrected control organoids, and SI_c1 and SI_c2 represent

clones derived from SI organoids corrected by cleavage with sgRNA1 and sgRNA2,

respectively. As seen in Figures A, C, and D10, the F508del patient organoids did not expand

their surface area, while the SI_c1 and SI_c2 without the CFTR inhibitor showed an increase in

the surface area. This means that the corrected CFTR genes in SI_c1 and SI_c2 worked

because of the increased surface area shown. You can also see that in Figures 2E and 2F,

where the error bars show a significant difference in surface area for both SI_c1 and SI_c2

when they have the CFTR inhibitor versus when they do not. This shows that without the CFTR

inhibitor, the surface area is significantly larger from the swelling. In Figures E and F for the

F508del, there is no significant difference between with and without the inhibitor of the CFTR

gene because there was no correction as this was the control group10.

The results of our study will be compared to Figure 4, which shows whether or not the

CFTR gene was corrected efficiently. Our study will use CRISPR/cas9 to cut out the faulty

CFTR gene and replace it with a correct copy of the CFTR gene. The study will also determine if

the CFTR gene was correctly replaced through experimentation. The difference will be that our

study will confirm if our CRISPR system worked by looking at the protein expression from a

western blot and DNA sequence from Sanger Sequencing.

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Figure 5. Shows the size, PDI and zeta potential of dry powder formulations. A-C shows the size

and PDI of TFFD formulations and D-F shows the zeta potential of TFFD formulations

containing mannitol, sucrose, and trehalose.

A second piece of literature we consulted was an article that looked into the best ways to

deliver gene editing modalities pulmonarily³. This study looked at gene editing dry powder

inhalation (DPI) formulations containing PEGylated chitosan/CRISPR-Cas9 nano complexes

prepared by thin-film freeze-drying (TFFD). Delivering our gene-editing complex through a

pulmonary system would allow for enhanced regional targeting and avoidance of biological

barriers that limit the efficiency of systemically (blood) administered therapeutics. Overall, this

study concluded that PEGylated chitosan/CRISPR-Cas9 nano complexes in dry powder are

suitable for inhalation and were best deployed using 3% of mannitol as the cryoprotectant

through TFFD. This DPI and TFFD process could be the delivery mechanism we employ to get

our CRISPR device into the patient’s lungs. Before even getting to the patient, we can even test

our method in a mouse model to study the safety and efficacy. While a mouse model would not

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be the exact match for how our pulmonary system works, it would give us a good early

indication of whether our therapeutic has a strong chance of success.

One technical limitation is the efficiency of nanoparticle delivery and endosomal escape.

Epithelial cells have barriers like the cell membrane that nanoparticles need to penetrate to

deliver the plasmid. Once inside the cells, the nanoparticle may get sequestered within

endosomes, limiting the release of the CRISPR/Cas9 into the cytoplasm, where it needs to act.

To overcome this, biocompatible materials like lipids can be used for our nanoparticle

fabrication, and incorporating components that promote endosomal escape, like pH-responsive

polymers or membrane-disruptive peptides into our nanoparticle formulation can facilitate the

release of CRISPR/cas9.

One biosafety concern we have regards the handling of CRISPR/cas9 genetically

modified cells. We can mitigate contamination when working with these plasmids by wearing the

appropriate PPE, including lab coats, gloves, and safety glasses, and in biosafety cabinets. We

will also ensure all waste products are autoclaved with any biological substance or

contamination. We will also ensure that any human cell lines we are working with have been

screened & cleared to be free of pathogenic viruses & bacteria. Another biosafety issue is

ensuring the CRISPR/cas9 system does not mutate the wrong gene. We ensure this won’t

happen by doing a triple check to ensure the correct gene is placed into the DNA. This is seen

in our project, which used Sanger Sequencing, PCR, and gel electrophoresis.

Proposed budget/cost:

Lab Supplies and Purpose Where they can be Approximate cost

Reagents needed purchased

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HSAEC1-KT cells Model delivery of ATCC $6,000

CRISPR/cas9 system https://www.atcc.org/

before moving to live products/crl-4050

organism trials.

PureLink™ HiPure Prep kit to isolate the ThermoFisher $377

Plasmid Filter plasmid DNA. https://www.thermofis

Midiprep Kit her.com/order/catalog

/product/K210014?IC

ID=cvc-pdna-midipre

p-c1b3

Transformation To test the efficiency iGEM registry n/a

Efficiency Kit of our competent

cells

pRSVCFTR CFTR expression Researcher who has n/a (quote needed)

plasmid vector these plasmids

https://www.ncbi.nlm.

nih.gov/pmc/articles/

PMC3776838/

AAV For transfections into VectorBuilder $2,099

the epithelial cells https://en.vectorbuild

er.com/products-servi

ces/service/adenoviru

s-packaging.html

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LNPs For encapsulation of VectorBuilder n/a (quote needed)

our plasmids https://en.vectorbuild

er.com/products-servi

ces/service/mRNA-so

lutions.html

pSB1AC3 Chloramphenicol iGEM registry n/a

Antibiotic Resistance

BBa_E0040 GFP fluorescence tag iGEM registry n/a

BBa_k2061002 sgRNA for plasmid iGEM registry n/a

BBa_K1218011 Cas9 enzyme iGEM registry n/a

Western blot kit To perform our ThermoFisher 149

Western plot

experiments

Proposed Timeline:

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Human Practices:

One stakeholder for our proposed project is patients and patient advocacy groups. From

their perspective, the proposed use of CRISPR/Cas9 for targeted gene editing offers hope for a

potential cure or significant improvement in managing CF. They may be eager for

advancements in genetic therapies that could alleviate symptoms, improve quality of life, and

extend the lifespan of individuals affected by CF. However, they may also have concerns about

the safety, accessibility, and ethical implications of such treatments and issues related to

affordability and equitable access to emerging therapies. Another stakeholder for our proposed

project could be regulatory authorities and policymakers at the national and international levels.

From their perspective, using CRISPR/Cas9 technology for gene editing raises important

regulatory and ethical considerations. They are responsible for evaluating the safety and

efficacy of new therapies and establishing guidelines and regulations to govern their

development, approval, and implementation. They must balance the potential benefits of

innovative genetic treatments with the need to ensure patient safety, ethical standards, and

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societal acceptance. They may also need to consider issues related to intellectual property

rights, healthcare infrastructure, and public funding for research and development in this field.

Cystic fibrosis (CF) is a genetic disorder that affects multiple organ systems, primarily

the lungs and digestive system, and is caused by mutations in the CFTR gene. Over the years,

significant progress has been made in understanding the molecular basis of CF and developing

treatments to manage its symptoms. However, there remains a critical need for more effective

therapies that address the underlying genetic cause of the disease. Recent advances in

genome editing technologies, particularly CRISPR/Cas9, have opened up new possibilities for

treating genetic disorders like CF. Researchers and organizations in the social sciences and

public health have been actively exploring the societal implications of these emerging genetic

therapies.

One key area of investigation is the ethical considerations surrounding using

CRISPR/Cas9 for human gene editing. Questions arise regarding the potential for unintended

off-target effects, the long-term safety of genetic modifications, and the implications of germline

editing for future generations13. Another societal concern is the accessibility and affordability of

CRISPR-based therapies13. As with many innovative medical treatments, there are challenges

related to equitable access to these therapies, particularly for marginalized and underserved

populations. Issues such as healthcare disparities, insurance coverage, and the high cost of

genetic therapies must be addressed to ensure that all individuals affected by CF have access

to potentially life-changing treatments.

Furthermore, public perception and attitudes toward genetic engineering and gene

editing significantly shape the regulatory landscape and public policy surrounding these

technologies. Public engagement efforts, education initiatives, and stakeholder dialogues are

essential for fostering informed decision-making and building trust in the ethical and responsible

use of CRISPR/Cas9 and other genome editing tools14.

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 While CRISPR/Cas9 holds great promise for treating genetic disorders like CF, it is

crucial to consider the broader societal implications and engage stakeholders such as patients

and authorities in discussions about the ethical, legal, social, and policy aspects of this rapidly

evolving field. Collaborative efforts between researchers, policymakers, advocacy groups, and

the public are essential for navigating the complex challenges and opportunities presented by

genome editing technologies in healthcare.

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References:
1.McBennett KA, Davis PB, Konstan MW. Increasing life expectancy in cystic fibrosis: Advances
and challenges. Pediatr Pulmonol. 2022;57 Suppl 1(Suppl 1):S5-S12. doi:10.1002/ppul.25733

2. Gonçalves GAR, Paiva RMA. Gene therapy: advances, challenges and perspectives. Einstein
(Sao Paulo). 2017;15(3):369-375. doi:10.1590/S1679-45082017RB4024

3. Zhang H, Zhang Y, Williams RO 3rd, Smyth HDC. Development of PEGylated

chitosan/CRISPR-Cas9 dry powders for pulmonary delivery via thin-film freeze-drying. Int J
Pharm. 2021;605:120831. doi:10.1016/j.ijpharm.2021.120831

4. Ong T, Ramsey BW. Cystic Fibrosis. Cystic Fibrosis: A Review. 2023;329(21):1859-1859.

doi:https://doi.org/10.1001/jama.2023.8120

5. Gill K, Fletch J. How Common Is Cystic fibrosis? Statistics and Risk Factors.
www.medicalnewstoday.com. Published September 20, 2023.
https://www.medicalnewstoday.com/articles/how-common-is-cystic-fibrosis#Learn-more

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and Challenges. Pediatric Pulmonology. 2021;57(S1). doi:https://doi.org/10.1002/ppul.25733

7. Allan KM, Farrow N, Donnelley M, Jaffe A, Waters SA. Treatment of Cystic Fibrosis: From
Gene- to Cell-Based Therapies. Frontiers in Pharmacology. 2021;12(12).
doi:https://doi.org/10.3389/fphar.2021.639475

8. Ridley K, Condren M. Elexacaftor-Tezacaftor-Ivacaftor: The First Triple-Combination Cystic

Fibrosis Transmembrane Conductance Regulator Modulating Therapy. The Journal of Pediatric
Pharmacology and Therapeutics : JPPT. 2020;25(3):192-197.
doi:https://doi.org/10.5863/1551-6776-25.3.192

9. Chen Q, Shen Y, Zheng J. A Review of Cystic Fibrosis: Basic and Clinical Aspects. Animal
Models and Experimental Medicine. 2021;4(3):220-232.
doi:https://doi.org/10.1002/ame2.12180

10.Schwank G, Koo BK, Sasselli V, et al. Functional repair of CFTR by CRISPR/Cas9 in

intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell. 2013;13(6):653-658.
doi:10.1016/j.stem.2013.11.002

11.Health Ethics & Governance (HEG). Human genome editing: recommendations.

https://www.who.int/publications/i/item/9789240030381. Published July 12, 2021.

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12.Human genome editing.; 2017. doi:10.17226/24623

13. Nhgri. Genome editing. Genome.gov.

https://www.genome.gov/dna-day/15-ways/genome-editing. Published March 13, 2019.

14. Shinwari ZK, Tanveer F, Khalil AT. Ethical Issues Regarding CRISPR Mediated Genome
Editing. Curr Issues Mol Biol. 2018;26:103-110. doi:10.21775/cimb.026.103

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