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American Journal of  Medical 
Science and Innovation (AJMSI) 

CRISPR-Cas9 Genomic Editing as an Innovation in the Management of  Sickle Cell
Disease: A Systematic Review

Solomon Musa1, Aloysius Obinna Ikwuka2*, Francis Chigozie Udeh2, Abdullahi Adobanyi Musa3, Ugo Collins Chukwuezie4

Volume 2 Issue 2, Year 2023
ISSN: 2836-8509 (Online)

DOI: https://doi.org/10.54536/ajmsi.v2i2.1760
https://journals.e-palli.com/home/index.php/ajmsi

Article Information ABSTRACT

Received: June 16, 2023

Accepted: July 08, 2023

Published: July 30, 2023

Genomic editing is a group of  technologies that scientists have used to alter an organism’s 
DNA. Of  the several genomic editing techniques, Clustered Regularly Interspaced Short 
Palindromic Repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) is well known. The 
CRISPR-Cas9 system is faster, cheaper, more accurate, more efficient than other genomic 
editing methods, and it is an adaptation from bacteria’s immune mechanism. Sickle cell 
diseases (SCDs) are a group of  monogenic diseases, and despite their high prevalence and 
chronic debilitating nature, they continue to have few therapeutic options available. The 
aim of  this study is to review existing literature and current clinical trials on CRISPR-Cas9 
genomic editing as an innovation in the management of  sickle cell disease (SCD), as well as 
the current state of  treatment for SCD. For this systematic review, PubMed, Google Scholar, 
African Journals Online (AJOL), and Clinicaltrial.gov articles published up to 6th October, 
2022 were searched. Searches for current clinical trials using CRISPR-Cas9 as intervention 
were conducted by using the search terms such as sickle cell disease, genomic editing, genetics, 
novel treatments, hematopoietic stem cell transplantation, gene therapy, and CRISPR-Ca9. 
Studies cited include meta-analyses, original research, prospective clinical trials, online 
abstracts, literature reviews, retrospective studies, case series, and scientific meetings. The 
primary search obtained 27,678 articles. Following a review of  titles and abstracts, a total of  
32 publications and 6 ongoing clinical trials were included in this systematic review based on 
the recent evidence-based management of  SCD. CRISPR-Cas9 genomic editing stands out 
as a novel, innovative technology which has the potential to cure SCD in children and adults 
with minimal side effects. Six clinical trials are ongoing with a huge potential for scaling up 
to Phases 3 and 4.

Keywords
CRISPR-Cas9, Genomic 
Editing, Innovation, 
Management, Sickle Cell 
Disease, Gene Therapy

1 Clinical Projects, Xcene Research, Ikeja, Nigeria
2 College of  Medicine and Health Sciences, American International University West Africa, Banjul, The Gambia
3 Department of  Family Medicine, Ahmadu Bello University Teaching Hospital, Zaria, Nigeria
4 Department of  Community Medicine, University of  Lagos, Surulere, Nigeria
* Corresponding author’s e-mail: aloysiussweet@yahoo.com

INTRODUCTION
Genetic medicine is a newer terminology for medical 
genetics and incorporates areas such as gene therapy. 
Recent advances in medical genetics are revealing etiologies 
for morphologic, endocrine, cardiovascular, pulmonary, 
ophthalmologic, renal, psychiatric, and dermatologic 
conditions (Ikwuka, 2023a).
Sickle cell disease (SCD) encompasses a group of  blood 
disorders resulting from inheriting two mutated copies of  
the β-globin gene (HBB) from both parents (Frangoul, 
2021). HBB, located on chromosome 11p15.5, encodes 
the β-chain of  hemoglobin (Onda, 2005). In normal 
circumstances, humans have three types of  hemoglobin: 
hemoglobin A (consisting of  2α and 2β chains), hemoglobin 
A2 (made up of  2α and 2δ chains), and hemoglobin F 
(HbF) composed of  2α and 2γ chains (Hall, 2020). In 
sickle cell anemia, the most prevalent form of  SCD, red 
blood cells (RBCs) contain an abnormal hemoglobin 
variant called hemoglobin S, wherein each of  the two 
β-chains has a specific mutation (an amino acid valine with 
codon GTG substitutes glutamic acid with codon GAG 
at position 6) (Hall, 2020). Thus, the genetics of  SCD (a 
hemoglobinopathy) is due to the substitution of  valine for 
glutamic acid at position 6 of  both β-globin polypeptide 
chains. Hemoglobin F persists until about 6 weeks of  
age. Thereafter, hemoglobin A persists throughout life. 

The mode of  inheritance of  SCD is autosomal recessive 
(Ikwuka, 2023b). 
Hemoglobin, a protein responsible for oxygen transport in 
red blood cells (RBCs), is synthesized during erythropoiesis 
(initiated in polychromatophil erythroblasts and continuing 
through the reticulocyte stage) (Hall, 2020). Erythropoiesis 
is stimulated and enhanced by erythropoietin, a substance 
synthesized by the kidneys. Results from different studies 
have shown that high levels of  blood pressure, glucose and 
lipid metabolic disorders, asymptomatic hyperuricemia, 
activation of  systemic immune inflammation and 
fibrogenesis, contribute to kidney damage (Ikwuka, 2015; 
Ikwuka, 2017a; Ikwuka, 2017c; Ikwuka, 2017d; Ikwuka, 
2017e; Ikwuka, 2018d; Ikwuka, 2019a; Ikwuka, 2019c; 
Ikwuka, 2022; Ikwuka, 2023d; Ikwuka, 2023e; Virstyuk, 
2016; Virstyuk, 2017a; Virstyuk, 2018a; Virstyuk, 2019; 
Virstyuk, 2021a; Virstyuk, 2021b), which contributes to 
anemia on account of  disturbed erythropoietin production. 
However, Dapagliflozin which is a Sodium-Glucose Linked 
Transporter 2 (SGLT-2) inhibitor and Liraglutide which is 
a Glucagon-like Peptide 1 Receptor Agonist (GLP-1 RA) 
have been found to increase the effectiveness of  treatment 
and improve the clinical course of  disease in patients 
with such comorbidities (Ikwuka, 2017b; Ikwuka, 2018a; 
Ikwuka, 2018b; Ikwuka, 2018c; Ikwuka, 2019b; Ikwuka, 
2021; Virstyuk, 2018b; Virstyuk, 2018c; Virstyuk, 2017b), 



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thereby reducing kidney damage, improve kidney functions, 
and reduce anemia. When hemoglobin S encounters 
low oxygen levels, it forms long crystals (sometimes 15 
micrometers in length) within RBCs, impeding their flow 
through narrow capillaries. The pointed ends of  these 
crystals can rupture the plasmalemma leading to sickle 
cell anemia (Hall, 2020). Major free radicals that are of  
physiological significance are superoxide anion, hydroxyl 
radical, and hydroperoxyl radical, while non-radical is 
hydrogen peroxide (Ikwuka, 2023c). Rauwolfia vomitoria 
has a neuroprotective ability at it elevates antioxidants and 
suppresses lipid peroxidation (Ekechi, 2023).
It is noteworthy that nearly two-thirds of  infants worldwide 
with HbSS or SCD are born in Nigeria, the Republic 
of  Congo, or India, where the childhood mortality rate 
associated with SCD remains alarmingly high (Piel, 2013). 
Symptoms and complications of  SCD typically manifest 
around 5 to 6 months of  age when fetal hemoglobin (HbF) 
synthesis significantly declines. These symptoms include 
severe anemia, episodes of  pain (referred to as sickle 
cell crisis), swelling in the hands and feet, and potential 
complications such as bacterial infections and stroke 
(Frangoul, 2021; Hall, 2020). Long-term pain can develop 
as individuals grow older, and the average life expectancy in 
developed countries ranges from 40 to 60 years (National 
Heart, Lung, and Blood Institute, 2015).
Newborn screening is the common diagnostic approach 
for identifying HbSS, and treatment options include 
penicillin (essential for children under five years with 
immature immune systems), folic acid supplementation, 
blood transfusions, vaccinations against encapsulated 
organisms, transcranial Doppler (TCD) screening 
to identify stroke risk in children (followed by blood 
transfusions, if  necessary), pain management, hydroxyurea, 
and intensive hospital-based care (Adams, 1998; Gaston, 
1986; Thornburg, 2012; WHO, 2011). Other diagnostic 
tests for SCD include sickling of  the red blood cells on 
a blood film which is induced by the addition of  sodium 
metabisulfite; and another test referred to as hemoglobin 
electrophoresis which detects abnormal hemoglobin 
forms (Ikwuka, 2023b). However, despite the significant 
need for effective treatment options for SCD patients, 
current treatments both traditional and newly developed, 
only ameliorate acute and chronic SCD manifestations 
without addressing the underlying cause. Hydroxyurea 
and long-term blood transfusions aim to prevent and treat 
complications associated with SCD. The recently approved 
crizanlizumab (Ataga, 2017) has shown a reduced 
incidence of  cellular adhesion and vaso-occlusive crisis 
in SCD patients, but it does not target the root cause of  
the disease or fully alleviate its manifestations (Frangoul, 
2021). Allogeneic hematopoietic stem cell transplantation 
(HSCT) remains the sole curative option for SCD, yet less 
than 20% of  eligible patients have a suitable HLA-matched 
donor (Baronciani, 2016; Eapen, 2019; Gluckman, 2017).
Further advances in the understanding of  the 
pathophysiology of  SCD contributed to the development 
of  the exciting and novel “Clustered Regularly Interspaced 

Short Palindromic Repeats (CRISPR)-associated protein 
9 (CRISPR-Cas9)” genomic editing therapy to cure 
the disease and its complications. Bacteriophages have 
the ability to infect bacteria by implanting its genetic 
material into the bacterial genome (Chaudhary, 2020). 
Thereafter, bacteria have a natural defense mechanism 
against bacteriophages, whereby on the first exposure to 
a bacteriophage they produce CRISPR sequence as a form 
of  genetic memory. CRISPR sequence is always found in 
association with the Cas9, a nuclease that can cleave the 
DNA. In subsequent exposure to a similar bacteriophage, 
the bacteria form guideRNA from the transcription of  
the CRISPR sequence. The guideRNA finds its target in 
the bacteriophage DNA, while the Cas9 cleaves the DNA 
(Chaudhary, 2020).
The advantage of  this system is that once the CRISPR 
system has cleaved the DNA, a DNA template carrying 
the desired sequence can join the cleaved end, thereby 
facilitating recombination and replacement of  the original 
sequence with the new version. The CRISPR-Cas9 nuclease 
system can be employed in cultured cells, including stem 
cells, as well as in fertilized eggs, enabling the generation of  
transgenic animals with targeted mutations. This genomic 
editing technique has been extensively studied in various 
organisms such as yeast, Drosophila, Zebrafish, plants, 
monkeys, and pigs, in addition to the bacteria from which 
the technique was originally derived from (Wen, 2017). 
In the case of  SCD, the CRISPR-Cas9 nuclease system 
is applied to hematopoietic stem and progenitor cells 
(HSPCs) at the erythroid-specific enhancer region of  the 
BCL11A locus on chromosome 2 (Uda, 2008). Normally, 
BCL11A encodes a transcription factor that inhibits HbF 
synthesis. The CRISPR-Cas9 nuclease system effectively 
suppresses BCL11A expression in erythroid-lineage cells, 
thereby restoring γ-globin synthesis and reactivating HbF 
production (Canver, 2015; Wu, 2019). Unlike previous 
genomic editing methods, CRISPR-Cas9 has the capacity 
to target multiple genes simultaneously, enabling the 
treatment of  not only diseases with point mutations but 
also those with polygenic mutations.
Researchers have recently realized that this system can 
be engineered to cleave DNA at precisely chosen loci, 
extending beyond viral DNA to any desired DNA 
sequence, simply by modifying the guideRNA to match 
the target (Chaudhary, 2020). In this systematic review, a 
comprehensive analysis of  the current state of  research 
on CRISPR-Cas9 for the treatment of  sickle cell disease 
(SCD) was conducted. By gathering information from 
multiple sources, evaluation of  the progress made with this 
innovative technology is determined, identified knowledge 
gaps for further research are checked, and the technology’s 
potential challenges and limitations are discussed.

METHODOLOGY
Search Strategy and Selection Criteria
This systematic review aimed to study all available literature 
on CRISPR-Cas9 genomic editing and its potential in the 
management of  SCD. It also sought to shed more light on 



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the subject matter (considering the fact this technology is 
novel) and its stage of  development is still in the clinical 
trials. A similar method of  literature search as described by 
Suwito, et al, 2023 was used (Suwito, 2023). The literature 
search was done from the following databases over a 
period of  2 weeks: PubMed (mostly used), Google Scholar, 
and African Journals Online (AJOL) using the following 
terms: CRISPR-Cas9, genomic editing, gene editing, sickle 
cell disease, hemoglobinopathies, sickle cell anemia, genetic 
therapy, systematic review, new therapy/novel intervention 
for sickle cell disease cure/treatment.

Data Sources and Search Engines
A literature search was done from the following databases: 
PubMed (mostly used), Google Scholar, and African 
Journals Online (AJOL). While the clinical trials search was 
done on ClinicalTrials.gov.

Inclusion and Exclusion Criteria
Included in this study were studies that investigated the 
use of  CRISPR-Cas9 genomic editing as a treatment for 
sickle cell disease, studies that included human participants 
or human cells/tissues, studies that provided data on the 
efficacy and/or safety of  CRISPR-Cas9 genomic editing 
for sickle cell disease, and studies that were published in 
English language within the past 10 years on gene therapy 
use in SCD, specifically CRISPR-Cas9. Excluded articles 
were studies not related to CRISPR-Cas9 genomic editing 
or sickle cell disease, studies that used animal or plant 
models only, studies not published in English, studies 

that did not provide data on the efficacy and/or safety of  
CRISPR-Cas9 genomic editing for sickle cell disease, and 
studies that had poor methodological quality or a high risk 
of  bias.

Quality Assessment of  Included Studies
The articles from the database search were reviewed to 
tailor them to the inclusion criteria. The abstracts of  the 
articles that met the inclusion criteria were reviewed for 
relevant keywords. The abstracts and the free complete 
articles i.e. manuscripts for the selected articles were then 
read, reviewed, and the information on each of  the key 
areas were summarized. This systematic review was carried 
out independently by four persons, to minimize errors. 
The summarized data were later compiled, reviewed, and 
discussed.

Data Extraction, Synthesis, and Results  
The following keywords were used to extract articles from 
database searches:

• “CRISPR-Cas9”
• “Genomic editing”
• “Sickle cell disease, hemoglobinopathies, and sickle       

cell anemia”
• “Genetic therapy and gene editing”
• “Systematic literature review or systematic review”
• “New therapy/novel intervention for sickle cell”.

Study Selection and Characteristics
The search for articles and abstracts was done using 

Figure 1: Steps of  article selection

keywords on the three major search engines (PubMed, 
Google Scholar, and AJOL). However, the mostly used 
search engine was PubMed due to its advanced features and 
its large repository of  articles. Study selection was based 
on articles, abstracts, or literature reviews which meet the 
inclusion criteria. Articles that were found under exclusion 

criteria were discarded. The diagram below illustrates how 
articles were selected.

RESULTS
The findings in this study are outlined in Tables 1 and 2.

Table 1: Summary of  Articles in the Literature Search
S/N Paper Title Abstract summary Study 

type
Outcome measured/
Summary of  conclusion

1 CRISPR-Cas9 Gene 
Editing for Sickle 
Cell Disease and 
β-Thalassemia (Frangoul, 
2021)

CRISPR-Cas9-targeting erythroid-
specific enhancers modified 80% 
of  the alleles at this locus in healthy 
donors.

Phase ½

• Allelic editing in bone marrow 
and blood
• ↑ in HbF
•Transfusion independence
• Elimination of  vaso-occlusive 
episodes in the patient with SCD



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2 A Review of  the 
Therapeutic Potential, 
Prospects, and 
Challenges of  CRISPR-
Cas9 Genome Editing 
in the Treatment of  
Sickle Cell Disease (SCD) 
(Chaudhary, 2020)

CRISPR-Cas9 is a potential 
therapeutic tool in the management 
of  SCD.

Review

• Through this review paper, 
the scope and possibilities of  
CRISPR-Cas9 as a potential 
therapeutic tool in the 
management of  SCD was 
analyzed

3 Therapeutic CRISPR-
Cas9 Genome Editing 
for Treating Sickle Cell 
Disease (Park, 2016)

Optimized CRISPR-Cas9 systems 
for genomic editing can be achieved 
in CD34+ cells.

Review

• Rates of  Non-Homologous 
End Joining (NHEJ) events
• Rates of  Homology Directed 
Repair (HDR) events
• Genome editing frequencies at 
both DNA and mRNA levels
• Expression of  globin and other 
erythroid markers
• Number and type of  colonies 
following induction of  
differentiation
• Genotype of  edited cells
• Translation of  edited β-globin 
protein and formation of  HbS

4 CRISPR-Cas9 Mediated 
Correction of  the Sickle 
Mutation in Human 
CD34+ cells (Hoban, 
2016a)

Targeted genomic editing 
technology can correct the SCD 
mutation of  the β-globin gene in 
hematopoietic stem cells.

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xperim

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• Gene modification rate
• Production of  wild type 
hemoglobin.

5 CRISPR-Cas9 to Induce 
Fetal Hemoglobin for the 
Treatment of  Sickle Cell 
Disease (Demirci, 2021)

Genomic editing is potentially 
a curative technique available 
to all individuals with 
-hemoglobinopathies, including 
SCD.

Review

• Fetal Hemoglobin (HbF) 
induction
• Editing of  transcriptional HbF 
silencers
• Modulating epigenetic 
intermediates that govern HbF 
expression

6 CRISPR-Cas9 for 
Sickle Cell Disease: 
Applications, Future 
Possibilities, and 
Challenges (Demirci, 
2019)

Genomic editing approach has 
proven valuable as a curative 
option.

Review

• Disease severity
• Mortality
• Morbidity

7 CRISPR-Cas9 Gene 
Editing for Curing Sickle 
Cell Disease (Park, 
2021a)

Ex vivo engineering of  autologous 
HSPCs followed by transplantation 
of  genetically modified cells 
potentially provides a permanent 
cure applicable to all patients 
regardless of  the availability of  
suitable donors and graft-vs-host 
reaction.

Review

• Severity of  pain
• End organ damage
• Early mortality

8 CRISPR-Cas9 Genomic 
Engineering: Trends in 
Medicine and Health 
(Zaib, 2022)

CRISPR-Cas9 technology offers 
the simplest, fastest, most versatile, 
reliable and precise method of  
genetic manipulation.

Review

• Genetic manipulation
• Removing sections of  the 
DNA sequence
• Adding sections of  the DNA 
sequence
• Altering sections of  the DNA 
sequence



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• Treatment of  various medical 
conditions including cancer, 
hepatitis B, cardiovascular 
diseases or even high cholesterol
• Minimization of  the off-target 
effects of  gene editing and 
incomplete matches between 
single guideRNA and genomic 
DNA by Cas9

9 CRISPR-Cas9: ANew 
and Promising Player in 
Gene Therapy (Xiao-Jie, 
2015)

CRISPR-Cas9 can be applied for 
therapeutic purposes in cell lines or 
animal models.

Review
 

• Gene therapy outcomes
• Correction of  causal mutations 
in monogenic disorders
• Rescue of  disease phenotypes
• Engineering of  pathogen 
genome for therapeutic purposes
• Induction of  protective or 
therapeutic mutations in host 
tissues
• Deactivation of  oncogenic virus
• Induction of  onco-suppressor 
expressions

10 Emerging Genetic 
Therapy for Sickle Cell 
Disease (Orkin, 2019)

Transcript factors that mediate 
silencing of  the γ-like fetal globin 
gene after birth have been identified 
and demonstrated to act as the 
β-globin promoters.

Review
 

• Efficacy of  genetic strategies to 
cure SCD
• Safety of  genetic approaches to 
cure SCD

11 Era of  Genomic 
Medicine: A Narrative 
Review on CRISPR 
Technology as a Potential 
Therapeutic Tool 
for Human Diseases 
(Kotagama, 2019)

The guideRNA can be modified to 
match a DNA sequence of  interest 
in the cell.

Review
 

• An insight with relation to a 
few of  the many diseases that 
are being tackled with the aid of  
the CRISPR-Cas9 mechanism 
and the trends, successes, and 
challenges of  this application as 
a gene therapy are discussed in 
this review

12 CRISPR-Cas9 Genome 
Editing in Human 
Hematopoietic Stem 
Cells (HSCs) (Bak, 2018)

Genomic editing via homologous 
recombination (HR) in human 
HSCs has the power to reveal 
gene-function relationships and 
potentially transform curative 
hematological gene and cell 
therapies.

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• Production of  HR targeted 
HSCs
• Enrichment of  HR targeted 
HSCs
• In vitro analysis of  HR targeted 
HSCs
• In vivo analysis of  HR targeted 
HSCs
• Manipulation of  genes for 
investigation of  gene functions 
during hematopoiesis
• Correction of  genetic mutations 
in HSC transplantation-based 
therapies for diseases such as 
SCD, β-Thalassemia, and Primary 
Immunodeficiencies

13 Efficacy of  CRISPR-
Based Gene Editing in 
a Sickle Cell Disease 
Patient as Measured 
through the Eye (Pinhas, 
2022)

Optical coherence tomography 
angiography can detect and measure 
micro-occlusive events within the 
retinal capillary bed before and 
after RBC exchange transfusion 
and following CRISPR-based gene 
editing.

Case report

• Micro occlusive events within 
the retinal capillary bed



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14 Current Sickle Cell 
Disease Gene Therapy 
Treatments: Literature 
Review (Ranadive, 2022)

The base editor shows promise in 
its ability to surpass many issues 
faced with both viral vectors and 
CRISPR-Cas9 in human trials.

Literature review

• Success of  lentiviral vectors in 
genotype correction and HbF 
induction
• Success of  CRISPR-Cas9 in 
genotype correction and HbF 
induction
• Success of  base editors in 
genotype correction and HbF 
induction

15 CRISPR-Cas9 Editing 
Induces High Rates of  
Unintended Large Gene 
Modifications in HSPCs 
from Patients with Sickle 
Cell Disease (Park, 
2021b)

Unintended on-target large 
deletions occur at high rates in 
gene-edited SCD HSPCs.

E
xperim

ental 

• Unintended gene modifications 
due to Cas9 induced Double 
Stranded Breaks (DSBs) in SCD 
HSPCs, including large deletions, 
insertions, and complex 
chromosomal arrangements

16 Automated Good 
Manufacturing Practice-
Compatible CRISPR-
Cas9 Editing of  
Hematopoietic Stem 
and Progenitor Cells for 
Clinical Treatment of  
β-Hemoglobinopathies 
(Urena-Bailen, 2023)

The enhancer of  the BCL11A 
gene is a CRISPR target in ongoing 
clinical trials for β-thalassemia and 
SCD treatment.

E
xperim

ental 

• Editing efficiency
• HbF resurgence

17 In vivo Selection for 
Corrected β-globin 
Alleles after CRISPR-
Cas9 Editing in Human 
Sickle Hematopoietic 
Stem Cells (HSCs) 
Enhances Therapeutic 
Potential (Magis, 2018)

Cas9-mediated gene editing in 
long-term engrafting human HSCs 
yields more than 20% correction 
of  the sickle mutation in long-term 
engrafting human HSCs.

E
xperim

ental 

• Percentage of  correction of  
the sickle mutation in long term 
engrafting human HSCs
• RNA sequence data to find 
cells carrying corrected SS globin 
alleles
• Efficiency of  editing with 
almost no off  target events

18 Multiplex CRISPR-
Cas9 Genomic Editing 
in Hematopoietic 
Stem Cells for Fetal 
Hemoglobin Reinduction 
Generates Chromosomal 
Translocations 
(Samuelson, 2021)

Genomic editing therapies targeting 
either the BCL11A erythroid 
enhancer or the HBG promoter 
are already proving successful in 
reinducing HbF.

E
xperim

ental 

• HbF reinduction
• Engraftment
• Lineage differentiation 
potential of  edited cells post 
xenotransplantation
• Chromosomal rearrangement 
events

19 CRISPR-Cas9: A 
Preclinical and Clinical 
Perspective for the 
Treatment of  Human 
Diseases (Sharma, 2021)

CRISPR-Cas9 is a promising 
genome-editing tool that has 
therapeutic potential against 
incurable genetic disorders by 
modifying their DNA sequences.

E
xperim

ental 

• Modulation of  predefined 
gene expression (upregulation or 
downregulation)

20 Genomic editing: A 
Perspective on the 
Application of  CRISPR-
Cas9 to Study Human 
Diseases (Review) 
(Rodriguez-Rodriguez, 
2019)

The CRISPR-Cas9 system can 
repair the damage caused to DNA.

Review
 

• The basic principles of  the 
CRISPR-Cas9 system are 
reviewed, as well as the strategies 
and modifications of  the 
enzyme Cas9 to eliminate the 
off-target cuts, and the different 
applications of  CRISPR-Cas9 
as a system for visualization and 
gene expression activation or 
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21 Use of  Genomic Editing 
Tools to Treat Sickle Cell 
Disease (Tasan, 2016)

The only existing curative treatment 
for SCD is based on allogeneic stem 
cell transplantation from healthy 
donors.

E
xperim

ental

• Discussion of  the three 
programmable nucleases that 
are commonly used for genomic 
editing purposes: Zinc Finger 
Nucleases (ZFNs), Transcription 
Activator-Like Effector 
Nucleases (TALENs) and 
CRISPR-Cas9

22 CRISPR-Cas9 β-globin 
Gene Targeting in 
Human Hematopoietic 
Stem Cells (Dever, 2016)

Ex vivo gene correction in 
patient-derived HSCs followed by 
autologous transplantation could be 
used to cure hemoglobinopathies.

Preclinical trial

• Homologous recombination at 
the HBB Gene in HSCs
• Efficiency of  correction of  the 
Glu6Val mutation responsible 
for SCD
• Expression of  adult β-globin 
(HbA) messenger RNA after 
differentiation into erythrocytes

23 A Systematic Review of  
Gene Editing Clinical 
Trials (Eshka, 2022)

There are promising phase-I and 
phase-II trials testing the safety 
and feasibility of  gene editing in 
different clinical settings.

Literature 
review

• Gene editing clinical trials
• Genetically engineered T-Cell 
therapies for cancer
• Virus infections
• Monogenic diseases

24 Genetic Treatment of  
a Molecular Disorder: 
Gene Therapy 
Approaches to Sickle Cell 
Disease (Hoban, 2016b)

The initial-retroviral vectors, next-
generation lentiviral vectors, and 
novel genomic engineering and 
gene regulation approaches share 
the goal of  preventing erythrocyte 
sickling.

Review
 

• Effective medical management 
for SCD
• Preventing erythrocyte sickling
• Clinical success

25 Efficient Ablation 
of  Genes in Human 
Hematopoietic Stem 
and Effector Cells using 
CRISPR-Cas9 (Mandal, 
2014)

CRISPR-Cas9 can efficiently ablate 
genes in HSPCs.

E
xperim

ental 

• Efficacy of  CRISPR-Cas9-
mediated genomic editing in 
primary human CD4+ T Cells 
and CD34+ HSPCs
• Gene deletion efficacy in 
CD4+ T Cells and CD34+ 
HSPCs
• Multilineage potential of  
HSPCs that had undergone 
genome editing with CRISPR-
Cas9
• Predicted on and off   target 
mutations via target capture 
sequencing in HSPCs
• Levels of  off  target mutagen

26 Selection-Free Genomic 
Editing of  the Sickle 
Mutation in Human 
Adult Hematopoietic 
Stem/Progenitor Cells 
(DeWitt, 2016)

A Cas9 RNP can mediate efficient 
hematopoietic stem cell genomic 
editing in human hematopoietic 
stem cells from sickle cell disease 
patients.

E
xperim

ental 

• Production of  normal 
hemoglobin
• Production of  HbS RNA and 
protein
• Production of  wild type 
hemoglobin

27 Genomic Editing for 
Sickle Cell Disease: A 
Little BCL11A Goes 
a Long Way (Hossain, 
2017)

CRISPR-Cas9 or ZFNs are useful 
tools to delete or replace sequences 
involved in the production of  
hemoglobin.

Review
 

• Feasibility of  genomic editing 
in HSPCs
• Ability of  CRISPR-Cas9 
or ZFNs to delete or replace 
sequences involved in the 
production of  hemoglobin



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28 Highly Efficient Editing 
of  the β-globin Gene 
in Patient-Derived 
Hematopoietic Stem and 
Progenitor Cells to Treat 
Sickle Cell Disease (Park, 
2019)

Gene-corrected sickle-cell HSPCs 
engrafted in vivo.

E
xperim

ental 

• Efficiency of  correcting the 
sickle mutation in the β-globin 
gene in HSPCs
• Reduction of  sickle cells in 
erythrocytes derived from gene 
edited cells
• Level of  normal adult 
hemoglobin (HbA) in 
erythrocytes derived from gene 
edited cells
• Engraftment of  gene-edited 
SCD HSPCs in Non-Obese 
Diabetic (NOD) SCID Gamma 
(NSG) mice

29 Cas9 Protein Delivery 
Non-Integrating 
LentiviralVectors for 
Gene Correction in 
Sickle Cell Disease 
(Uchida, 2021)

The Cas9 protein delivery non-
integrating lentiviral all-in-one 
system efficiently corrected 
the SCD mutation in the 
endogenous S-globin gene without 
electroporation.

E
xperim

ental 

• Efficiency of  correction 
of  the SCD mutation in the 
endogenous β-globin genes
• Protein level of  the corrected 
β-globin genes

30 Application of  CRISPR-
Cas9 Genomic Editing 
in Genetic Disorders: A 
Systematic Review Up to 
Date (Pandey, 2017)

CRISPR-Cas9 system has been 
used from last few years in the field 
of  biomedical research.

Review
 

• Genomic editing technologies 
over the past few years is 
providing fast and effective 
tool to precisely manipulate the 
genome at specific locations.

31 Development of  
β-globin Gene 
Correction in Human 
Hematopoietic Stem 
Cells as a Potential 
Durable Treatment 
for Sickle Cell Disease 
(Lattanzi, 2021)

Ex vivo β-globin gene correction in 
autologous patient-derived HSPCs 
may potentially provide a curative 
treatment for SCD.

Phase 1/2

• Gene correction
• Genotoxicity
• Tumorigenicity
• Multilineage engraftment
• Abnormal hematopoiesis
• Toxicology

32 Combination of  
Lentiviral and Genomic 
Editing Technologies for 
the Treatment of  Sickle 
Cell Disease (Ramadier, 
2022)

Transduced cells from sickle cell 
patients were transduced with 
lentiviral vectors expressing AS3 
and a guideRNA either targeting 
the endogenous β-globin gene or 
regions involved in HbF silencing.

E
xperim

ental 

• Clinical benefit in SCD patients
• Vector Copy Number (VCN)
• Anti-sickling hemoglobins
• Rescue of  the SCD phenotype
• Genotoxicity risk

Table 2: Current Clinical Trials on CRISPR-Cas 9 (from Clinicaltrial.gov, assessed on April 5th, 2023)
S/N Study Title Conditions Interventions Locations (First 3)
1 A Safety and 

Efficacy Study 
Evaluating CTX001 
in Subjects with 
Severe Sickle Cell 
Disease

• Sickle cell disease
•Hematological 
diseases
•Hemoglobino-
pathies

• Biological: CTX001
• Phase 1/2/3 study
• 45 estimated 
participants

1. Lucille Packard Children’s Hospital 
of  Stanford University, Palo Alto, 
California, United States.
2. Ann & Robert Lurie Children’s 
Hospital of  Chicago, Chicago, Illinois, 
United States.
3. University of  Illinois at Chicago 
Hospitals and Health Systems, 
Chicago, Illinois, United States.



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2 Evaluation of  
Efficacy and 
Safety of  a Single 
Dose of  CTX001 
in Participants 
with Transfusion-
Dependent Beta 
Thalassemia and 
Severe Sickle Cell 
Disease

• β-Thalassemia
• Thalassemia
• Hematologic 
disease

• Biological: CTX001
• Phase 3
• 12 estimated 
participants

1.Columbia University Medical 
Center, New York, United States.
2. Atrium Health Levine Children’s 
Hospital,Charlotte, North Carolina, 
United States.
3. SCRI at the Children’s Hospital 
at TriStar Centennial, Nashville, 
Tennessee, United States.

3 Evaluation of  
Safety and Efficacy 
of  CTX001 
in Pediatric 
Participants with 
severe SCD

• SCD
• Hydroxyurea 
failure
•Hydroxyurea 
intolerance

• Biological: CTX001
• Phase 3
• 12 estimated 
participants

1. Children’s Hospital of  Philadelphia, 
Philadelphia, Pennsylvania, United 
States.
2. St. Jude Children’s Research 
Hospital, Memphis, Tennessee, 
United States.
3. The Children’s Hospital at TriStar 
Centennial Medical Center/ Sarah 
Cannon Center for Blood Cancers.

4 Transplantation of  
CRISPR- Modified 
Hematopoietic 
Progenitor Stem 
Cells (CRISPR-SCD 
001) in Patients with 
Severe Sickle Cell 
Disease

• SCD • Drug: CRISPR_
SCD001
• Phase 1/2
• 9 estimated 
participants

1. University of  California, Los 
Angeles, California, United States.
2. UCSF Benioff  Children’s Hospital, 
Oakland, California, United States.

5 Gene Correction in 
autologous CD34+ 
Hematopoietic stem 
cells (HbS to HbA) 
to treat severe SCD

• SCD • Genetic: GPH101 
Drug Product
• Phase 1/2
• 15 estimated 
participants

1.University of  Alabama, 
Birmingham, Alabama, United States.
2. Lucile Packard Children’s Hospital, 
Palo Alto, California, United States.
3.Washington University, Saint Louis, 
Missouri, United States.

6 A Long-Term 
Follow-up Study 
in Patients Who 
Received CTX001

• β-Thalassemia
• Thalassemia
• SCD

• Biological: CTX001
• Cohort Study
• 114 estimated 
participants

1. Columbia University Medical 
Center (21+ years), New York, United 
States.
2. Columbia University Medical 
Center, New York, United States.
3. Children’s Hospital of  Philadelphia, 
Pennsylvania, United States.

DISCUSSION
This systematic literature review provides an overview 
of  CRISPR-Cas9 genomic editing and its application to 
sickle cell disease (SCD), while also addressing the ethical 
implications associated with this technology in SCD 
management. Despite the wide use of  CRISPR-Cas9 as a 
mature genomic editing tool, therapeutic applications still 
face challenges such as off-target effects, complex in vivo 
Cas9 protein delivery, low gene editing efficiency, and 
packaging issues. To become an ideal delivery method 
for therapeutics, CRISPR-Cas9 strategies should exhibit 
high delivery efficiency, precise targeting ability, and 
ease of  mass production. However, current approaches 
in this field are far from achieving this desired level of  
performance (Guo, 2022). While there is significant 
literature on why SCD is a suitable candidate for 

CRISPR-Cas9, less attention has been given to the ethical 
implications of  including SCD in CRISPR-Cas9 research.
In addition, the implications of  CRISPR-Cas9 for sickle 
cell disease have significant consequences for clinical 
practice and policy. The following points highlight some 
of  the potential implications:

Improved Outcomes
CRISPR-Cas9 holds the potential to cure SCD by 
correcting the underlying genetic mutation. This 
breakthrough could lead to improved outcomes for 
patients, including reduced pain, enhanced quality of  life, 
and increased lifespan.

Reduced Healthcare Costs
SCD treatment can be expensive, and the use of  CRISPR-



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Cas9 may reduce healthcare costs by offering a curative 
approach rather than merely managing symptoms.

Ethical Considerations
The use of  CRISPR-Cas9 in humans raises ethical 
concerns regarding safety and the possibility of  unintended 
consequences. The development of  policies is necessary 
to ensure the ethical and reliable application of  CRISPR-
Cas9.

Access to Treatment
Issues related to access to CRISPR-Cas9 treatment for 
SCD patients may arise, particularly in low- and middle-
income countries. Policies should be developed to ensure 
equitable access to the benefits of  this technology for all 
patients in need.

CONCLUSION
After analyzing the available evidence on the use of  
CRISPR-Cas9 for the management of  SCD, it can be 
concluded that this technology is novel and shows promise 
as a potential therapeutic option for the condition. Studies 
have demonstrated the successful correction of  the genetic 
mutation responsible for SCD in clinical settings. One of  
the main challenges of  this technology is the delivery of  
the CRISPR-Cas9 system to the bone marrow, where the 
hematopoietic stem cells reside. The off-target effects of  
the CRISPR-Cas9 system also need to be further studied 
and minimized to ensure the safety of  the treatment.
Despite these challenges, the potential benefits of  
CRISPR-Cas9 for SCD cannot be neglected. The ability to 
correct the genetic mutation responsible for the condition 
offers a potentially curative approach to the disease. 
Overall, further research and clinical trials are necessary 
to fully evaluate the safety and efficacy of  CRISPR-Cas9 
as a therapeutic option for SCD. Nevertheless, the current 
evidence suggests that CRISPR-Cas9 has the potential to 
revolutionize the treatment of  this debilitating disease by 
offering a curative option.

Conflict of  Interest
The authors hereby declare no conflict of  interest in 
conducting this research, and in publishing this manuscript.

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