CRISPR/Cas9 Based Gene Therapy

Ece Odabaşı^T., Gülnur Uzun^E.

The gene, which is the basic unit of heredity in living things, structurally contains the untranslated region (UTR), enhancer or silencer, and promoter areas at the 5′ and 3′ ends, and coding exons and non-coding introns between them. Gene expression is regulated transcriptionally by messenger RNAs (mRNA) formed as a result of alternative splicing mechanism¹. Gene therapy tries to prevent genetic diseases by activating an inactivated gene caused by genetic mutations or by preventing the expression of unwanted genes². Gene editing technologies provide an entirely new modality for treatments based on precise modification of human genome sequences^3,4.

Clustered regulatory spaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) are novel and proficient RNA-guided endonuclease-based system adapted from the naturally occurring bacterial immune system⁵ and has been used since 2012 as a genome editing tool as well as gene therapy for the treatment of cancer, infections, and genetic diseases^6-9. In this review, future aspects and potential applications of CRISPR/Cas9 technology, which is necessary for the treatment of genetically interacting diseases, will be discussed.

CRISPR/Cas9 system

CRISPR/Cas9 technology, when introduced into a cell, recognizes the conserved sequences of PAM (protospacer adjacent motif) next to the target DNA sequences, single guide RNA (sgRNA), and sgRNA positions the Cas enzyme on the targeted DNA sequence. sgRNA includes trans-activating CRISPR RNA (tracrRNA) and CRISPR RNA (crRNA). TracrRNA binds to the Cas9 protein, which hybridizes with crRNA and forms the CRISPR-Cas9/sgRNA complex to edit genome sequences. Upon this interaction, endonuclease domains cleave both DNA strands three bases upstream of the PAM sequence. The Cas9-sgRNA complex dissolves the double-stranded DNA (dsDNA) so that the complementary sequence in the sgRNA is attached to one of the DNA strands, and Cas nuclease cuts the double strand of DNA, creating a double-strand break (DSB) 10,11. In recent years, the use of CRISPR/Cas strategies, including knock-in (gene insertion, KI), knock-out (gene knockout, KO), base editing (BE), and primary editing (PE) in variations that cause genetic diseases, has brought a groundbreaking view to gene therapy ^2,12(Figure 1).

Figure 1. An overview of the four main techniques of CRISPR/Cas gene therapy². a) Gene knock out. b) Gene knock-in/replacement. c) Base arrangement d) Primary arrangement.

1. DSB-mediated Gene Editing

There are many coding and non-coding genes in organisms. Of the non-coding genes, most of the long noncoding RNA (long ncRNA) and microRNA (miRNA), known as genes that play a role in cell physiology and regulate gene expression, serve as tumor suppressors and proto-oncogenes^13,14. The KO method involves introducing CRISPR/Cas9 into the cell for genetic manipulation, and various repair mechanisms in the cell are activated after DSBs are formed in the DNA. In the non-homologous end-joining (NHEJ) mechanism, protein factors recombine directly or by incorporating nucleotide deletions or insertions into DNA strands; however, the open reading frame (ORF) of the gene is changing, so it is possible to silence or delete an unwanted gene by creating KO^15,16.

A repair mechanism called Homology Directed Recombination (HDR) is used in the KI method. It differs from NHEJ in that it requires a template to repair DSBs, which can be endogenous or exogenous, inserted into its genome, or composed of homologous DNA sequences. Duplication occurs when cells use homologous sequences as DNA templates. Exogenous DNA templates, on the other hand, cause horizontal gene transfer so that an undesired region of the genome can be converted into a determined gene sequence¹⁷. NHEJ and HDR mechanisms often result in small insertions/deletions (indels), out-of-frame mutations, early stop codons, or loss of function^18,19. The low success frequency of KO and KI methods led to the development of BE and PE methods¹⁷.

2. Base Editing

Mutations called single nucleotide polymorphism (SNP) resulting from a single base change constitute the proportionally largest proportion of human genetic diseases²⁰. Base editing is an approach based on irreversibly changing one base pair to another base pair at the genomic target locus and does not require DSB, HDR, or donor DNA²¹. In this way, errors that occur during DNA repair are minimized. DNA base modifiers are formed by a catalytically inactivated Cas nuclease enzyme (dead Cas9/dCas9) or a deaminase fused nickase Cas9 (nCas9). After cleavage at the target locus, a deaminase binds to the opposite DNA strand and modifies the bases. Base editing tools²¹ (Figure 2), which are divided into two classes as cytosine base editors (cytosine base editor, CBE)²² and adenine base editors (ABE)²³, are frequently used as they have a relatively low-risk ratio compared to CRISPR-Cas systems inducing DSBs, although they show indel risk^24,25.

Figure 2. Cytosine Base Regulator (CBE) and Adenine Base Regulator (ABE)²¹. CBE is able to bind to target DNA under the direction of Cas9. Apoliprotein mRNA cytidine deaminase 1 (APOBEC1) catalyzes the hydrolytic deamination of cytosine (C) in DNA and RNA to form uracil (U). It is then converted to thymine (T) by DNA repair mechanisms. When Cas9 directs ABE to the desired region in the target DNA, adenine (A) is converted to inosine (I) by adenine deaminase (ecTadA). It is converted to guanine (G) by DNA repair mechanisms.

2.1. Cytosine Base Regulators (CBE)

It mainly consists of CBEs, Cas nuclease, and activation-induced cytidine deaminase, AID (APOBEC/AID). Advanced top models have been designed since the first base editor (BE1), which was developed by combining APOBEC1 with the N-terminus of the dCas9 nuclease²⁶.  The BE-sgRNA complex binds to the target genomic DNA. This results in an RNA-DNA hybrid and a single-stranded DNA loop not bound to sgRNA around the target site. ssDNA can then be used for rAPOBEC1-catalyzed deamination of cytosine within the five nucleotide window. The second developed cytosine base regulator (CBE2) was developed by the fusion of the uracil DNA glycosylase inhibitor (UGI) with BE1. CBE3 is formed by the recovery of the histidine amino acid (H840, HNH catalytic domain) at position 840 in dCas9^23-25.

2.2. Adenine Base Regulators (ABE)

For the first ABE designed with theantibiotic resistance complement approach, TadA mutants, and defective antibiotic resistance genes were generated in Escherichia coli cells. TadA-dCas9 fusion corrected the targeted A base in the mutant chloramphenicol resistance gene. ABE1 is XTEN to the N-terminus of nCas9 in the TadA variant (TadA*) (16 amino acids linked are used in BE3); It was formed by the addition of the nuclear localization signal (NLS) to the C terminal (TadA*-XTEN-nCas9-NLS). It can change to A-G or A-T to G-C by adenosine deamination²⁷.

3. Prime Editing

PE, developed by Anzalone et al., is a gene editing method that can perform targeted small indels and base changes, does not cause DSBs that occur in classical CRISPR methods, and does not require a donor DNA template^28,29. PEs use a primary editing guide RNA (pegRNA) hybridized to the target DNA region and a nCas9-fused reverse transcriptase (RT) enzyme³⁰. The 5′ end of the pegRNA binds to the primer binding sequence (PBS) of the DNA, showing the non-complementary sequence. The PAM-containing chain is nicked by Cas9 and forms a primer for nCas9-dependent RT. Thanks to RT, the nicked PAM sequence uses the interior of the pegRNA as a template and makes the target region programmable (Figure 3) ³¹. With nCas9, targeted indels and off-target effects can be predicted in the genome. PE has the ability to regulate target frameshift or indel mutations with much higher efficiency than HDR³². Three major regulatory (PE) systems have been established and tested in human cells. The first system (PE1) was constructed with a fusion of Cas9(H840A) nicase and wild-type Moloney murine leukemia virus RT enzyme. In the second system (PE2), five specific mutations were added to M-MLV RT to increase the stability, affinity, and processing of DNA-RNA substrates²⁸. 

While L603W, D200N, and 330P mutations increase the number of transversions applied at high temperatures and RT activity, RT thermostability and the bond of the RT-template-PBS complex are strengthened thanks to two mutations called W313F and T306K. The PE2 system consists of the fusion of pentamutant RT with nCas9. PE2 showed a 5.1-fold improvement in the efficiency of the main regulation point mutation compared to PE1³³. The PE3 system differs from PE2 in that it cuts the unedited strand away from the pegRNA target and uses an additional gRNA (sgRNA) for this. Thus, regulation efficiency increases^34,35.

Figure 3. Primary Arrangement³¹.  PEs consist of an nCas9 fused reverse transcriptase (RT) and a pegRNA. The pegRNA has two sequences that direct nCas9 to the target sequence and contain the desired changes. nCas9 nicks the PAM sequence after binding to PBS. Thanks to PE, all transversion/transition mutations and small indels can be edited.

4. Primary Regulation and Base Regulation Application Areas

PEs have been tested in post-mitotic mouse cortical neurons, multiple human cell lines²⁹, mouse embryos³⁶, human pluripotent stem cells (iPSC)³⁷, rice and wheat protoplasts^30,38-40 and supported with varying efficiencies. In short, it has been shown that in DGAT1 (Diacylglycerol O-acyltransferase 1) deficiency in Wilson’s disease, which is known as copper accumulation in the organs, the in-frame deletions and frameshift mutations that cause the disease are corrected by PE⁴¹. Rousseau et al., using PE, aimed to prevent the development of the disease by creating the Ala673Thr variant in the APP gene, which has an important role in Alzheimer’s disease⁴². Thanks to BE, fetal hemoglobin (alpha2gamma2) of transcription factor B-cell lymphoma/leukemia 11A (BCL11A) was suppressed in adult-stage erythroid cells, hereby treating the core sequence β-thalassemia-induced disorder⁴¹. The disease phenotype was corrected by transforming GGT>CCC in the β-thalassemia mouse model with IVS-II-654 mutation with PE3⁴³. Krishnamurthy et al. developed a tool to repair rare cystic fibrosis transmembrane conductance regulator (CFTR) mutations by delivering BE ribonucleoproteins (RNP) to human airway epithelial cells; thus, a viable gene editing strategy for cystic fibrosis disease has been demonstrated⁴⁴. CRISPR/Cas9 technology holds great promise as a treatment for existing chronic viral infections such as human immunodeficiency virus (HIV), human papillomavirus (HPV), hepatitis B virus (HBV), and herpesvirus^45-47. Studies on CRISPR–Cas9 applications have also been conducted in different types of cancer treatment^48-53. Today, important experimental studies are carried out on monogenic diseases^54-57 and antibiotic resistance in model organisms⁵⁸.

In conclusion, CRISPR-Cas-based gene therapy has been a promising approach for the treatment of genetic diseases. CRISPR-Cas9 technology has been used in a wide range of domains, from fundamental biology to cancer therapy, since it is a potent, low-cost, and easy-to-create genome editing tool. Research on the use of this system in the treatment of various diseases continues rapidly.

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