Navigating the CRISPR/Cas Landscape for Enhanced Diagnosis and Treatment of Wilson’s Disease

Figures

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  Figure 1. Copper uptake and homeostasis pathway in the human body. Copper is absorbed mainly in the duodenum and partly in the stomach via proteins such as high-affinity copper uptake protein 1 (CTR1) and divalent metal-ion transporter 1 (DMT1). Before transportation, cupric ions (Cu2+) are reduced to cuprous ions (Cu+) with the assistance of six transmembrane epithelial antigen of the prostate (STEAP) reductase, ascorbate, or duodenal cytochrome b (DCYTB). Upon absorption by and entry into an enterocyte, copper is transported by key copper chaperones, predominantly cytochrome c oxidase copper chaperone (COX17), superoxide dismutase (CCS), and antioxidant-1 (ATOX1). COX17 transfers copper to mitochondrial proteins SCO1 and SCO2, which are crucial for cytochrome c oxidase biogenesis. CCS delivers copper to superoxide dismutase (SOD1), which is a major antioxidant maintaining cytoplasmic ROS homeostasis. Copper is transferred by ATOX1 to the trans-Golgi network (TGN), where it interacts with copper-transporting P-type ATPase 1 (ATP7A) and P-type ATPase 2 (ATP7B). The ATP7A facilitates the delivery of Cu+ to copper-dependent enzymes such as peptidyl-α-monooxygenase, tyrosinase, and lysyl…

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  Figure 2. The structure of ATP7B. The A-domain has a TGEA motif, the P-domain has a DKTGT motif, and the N-domain has a TGDN and a SEPHL motif. There are six metal-binding domains (MBDs) containing the core conserved sequence GMXCXXC at the N-terminus located intracellularly, each of which can bind to one Cu+ ion. Among the transmembrane domains (TMDs), TMD6 has a CPC motif, which makes it characteristic of heavy metal-transporting P-type ATPase. The illustration was created using BioRender.com.

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  Figure 3. Prevalent mutation variants on the ATP7B gene that induce Wilson’s disease (WD). The most common mutations in the ATP7B genomic DNA causing WD are highlighted in blue (p.R778L/c.2333G>T; Asia) and yellow (p.H1069Q/c.3207C>A; Europe and North America). No predominant mutation was found to be associated with South America [28]. The p.H1069Q mutation occurs in the SEHPL motif in the N-domain, while the p.R778L mutation affects transmembrane transportation activity. The red dots indicate the locations of the mutation variants shown in Table 1.

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  Figure 4. Schematics of the mechanisms of the CRISPR/Cas system types. The CRISPR/Cas system is classified into two classes based on the effector molecules involved. Class I is characterized by multi-unit effectors, while Class II exhibits a single effector. Each class is further divided into three types distinguished by their catalytic domain and target nucleotide specificity. Type I, represented by Cas3, forms a cascade complex with Cas6, Cas7, Cas5, Cas11, and Cas8. Upon crRNA binding, Cas3 generates a single-strand nick on the unwinding target DNA. Utilizing the Cas10-Cmr/Cms complex, the Type III system degrades nascent RNA before enzymatically cleaving complementary DNA. Type IV comprises csf1, csf2, csf3, an endoribonuclease (csf5), and a helicase (dinG), although its mechanism is not completely understood. The most widely used Type II system employs a single effector, Cas9, which induces a double-strand break (DSB) to target the double-stranded DNA (dsDNA) alongside the guide RNA (crRNA and tracrRNA). Types V and VI possess both target and collateral cleavage activities. Cas12 functions in conjunction with crRNA to generate staggered dsDNA breaks and nonspecifically cleaves the…

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  Figure 5. CRISPR/Cas system-based diagnosis of ATP7B mutations. (A) Diagnosing ATP7B mutations in WD patients depends on the specific type of mutation in the ATP7B gene and the type of nucleic acid (genomic DNA (gDNA) or mRNA) extracted from the patient. The red star represents the mutation point. The appropriate Cas protein must be selected and applied with these factors in mind. (B) A simple example of how the CRISPR/Cas system could potentially be used for diagnosing four distinct ATP7B mutations. The yellow highlight represents PAM sequences for SpCas9 (5′-NGG) and AsCas12a (5′-TTTN). The blue highlight represents the crRNA sequence in sgRNA to recognize the target sequence. The asterisk on the amino acid mutation means the stop codon. (C) The figure shows the locations in the genomic DNA (gDNA) and cDNA where the c.2072G>T (p.G691V) and c.2108G>A (p.C703Y) mutations can occur. The yellow highlight represents PAM sequences for SpCas9. The blue highlight represents potential sgRNA candidates for SpCas9, which are in proximity to the p.G691V and p.C703Y mutations. (D) The RGEN-RFLP method distinguishes between WT and mutant sequences by cutting the WT using sgRNA that matches the WT…

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  Figure 6. Schematics of the mechanism of four different CRISPR/Cas system-based treatment strategies. (A) To induce a homology-directed repair (HDR) event mediated by the Cas9 protein, it is necessary to provide donor DNA, which should include the wild-type sequence of the ATP7B gene. The Cas9 nuclease then induces a DSB in the vicinity of the mutation on the ATP7B gene. It is then necessary to select a suitable repair pathway for the treated cell. (B) Adenosine deaminase within the adenine base editor (ABE) system catalyzes the conversion of adenine (A) to inosine (I) on the strand not bound by the sgRNA. The nCas9 enzyme induces a nick in the DNA, initiating the DNA repair mechanism. Subsequently, during replication of the target DNA, a conversion of a thymine (T)/A base pair to a cytosine (C)/guanine (G) base pair is accomplished. (C) The cytosine base editing (CBE) is similar to that of ABE. In this case, a G/C-to-A/T base pair conversion is achieved via a uracil (U)-containing intermediate. Uracil glycosylase inhibitor (UGI) provides stability to the U/G base pair. (D) Unlike base editing, the prime-editing system utilizes a specific nCas9 variant with the H840A mutation, which…