Development of HEK293T cell reference materials for β-thalassemia genetic testing using prime editing

Introduction

β-Thalassemias are a group of heterogeneous autosomal recessive hereditary blood disorders characterized by reduced (β+) or absent (β0) synthesis of the β-globin chain [1]. It is estimated that there are 80 to 90 million β-thalassemia carriers worldwide, and approximately 68,000 children born with various thalassemia syndromes each year [2], [3], [4]. Over 400 hemoglobin-β gene (HBB) mutations have been identified to date (Ithanet database, https://www.ithanet.eu/), including single-nucleotide variants (SNVs), small insertions, or deletions, which affect gene transcription, RNA processing, and mRNA translation [5]. Clinical severity could be affected by the mutation type. Patients who are homozygous or compound heterozygous for these mutations develop β-thalassemia major and require lifelong blood transfusions and iron chelation therapy [3]. Individuals with β-thalassemia traits have a 25 % risk of having children affected with thalassemia major if their partner is also a carrier. Nowadays, HBB mutations can be detected by laboratory molecular analysis based on reverse dot-blot hybridization (RDB) analysis, melting curve analysis, allele-specific amplification or sequence analysis [6], 7]. Accurate molecular analysis is essential to meet the high demand of population-based screening, genetic counseling, and prenatal diagnosis of β-thalassemia.

Reference materials are essential for validating and ensuring the accuracy and reliability of genetic testing across different assays, platforms, and laboratories in clinical genetic testing [8]. They also support external quality assessment programs, testing development and validation, as well as proficiency testing and training [9]. Therefore, the use of reference materials for laboratory quality assurance is mandated by regulatory requirements and professional guidelines. Traditionally, plasmids, pseudoviruses, residual patient samples or commercial cell lines are used as reference materials [10]. Ideally, reference materials should mimic patient samples and encompass all variant alleles or mutation types targeted by the assay [11]. Genomic DNA sourced from cell samples, preferably derived from residual patient specimens or immortalized B lymphoblastoid cell lines, can be used for clinical β-thalassemia molecular analysis [12]. However, collecting enough peripheral blood samples from corresponding mutation carriers can be challenging, especially for rare or homozygous HBB mutations. Additionally, immortalized B lymphoblastoid cell lines have greater production cost, longer production cycle, longer population doubling time and lower yield compared to conventional HEK293T cells [13]. To address the need for publicly available, renewable, and characterized genomic DNA reference materials for β-thalassemia, we proposed using programmable gene-editing tools to efficiently establish stable HEK293T cell lines with HBB mutations causing β-thalassemia.

Prime editing (PE), established by David Liu’s research group, introduces a novel two-component genome editing platform that enables targeted gene correction, precise substitution, insertion, or deletion, rather than gene disruption [14]. Unlike early gene editing techniques such as CRISPR-Cas9, PE operates without the requirement for double-strand breaks (DSBs) or homologous directed recombination (HDR). It achieves higher editing efficiency while avoiding adverse outcomes associated with DSBs, such as unnecessary mixtures of insertions and deletions (indels) at the target site and HDR-mediated correction inefficiency [15]. Additionally, compared to cytosine base editors (CBEs) and adenine base editors (ABEs), PE offers a broader range of editing types, including multiple-site editing, DNA fragment insertion, and deletion [16]. The prime editor 3 (PE3) system reduces the risk of cellular mismatch repair by incorporating an additional sgRNA that guides the editor to nick the unedited DNA strand, thus significantly improving editing efficiency [17]. Its applicability in gene editing has been validated in various cell lines including HEK293T, K562, HeLa cells [18], stem Leydig cells [19], and mice [20].

Since PE systems offer a remarkably versatile and precise gene editing platform capable of potentially generating all mutations of the HBB in β-thalassemia, we aimed to generate a panel of HEK293T cell lines with distinct HBB mutations using the PE3 system. We successfully established 12 HEK293T cell lines with various heterozygous or homozygous HBB mutations (including SNVs, insertions, and deletions). These new cell lines were thoroughly characterized by various methods, confirming their suitability as reference materials for β-thalassemia genetic testing.

Materials and methods

Results

Construction of novel HBB mutation HEK293T cell lines by PE3 system

We employed the PE3 system to establish HBB mutations in HEK293T cells. As Illustrated in Figure 1A, PE3 systems consist of a prime editor, combining a Cas9 nickase fused with an M-MLV reverse transcriptase (RT), along with a prime editing guide RNA (pegRNA) and an additional nicking sgRNA. The pegRNA comprises a spacer, scaffold, RT template (RTT), and primer-binding site (PBS). Upon delivery into cells, the pegRNA spacer guides the prime editor to the target site, where Cas9 nicks one DNA strand, exposing a 3′ end. The PBS then anneals to this end, serving as a substrate for RT to synthesize the desired edit encoded by the RTT. In the PE3 system, an additional sgRNA nicks the nonedited DNA strand, enhancing editing efficiency. Subsequently, the newly synthesized DNA strand is integrated into genomic DNA by cellular repair machinery to achieve correction.

Figure 1:

Construction of HBB mutant cell lines using the PE3 system. (A) Architecture of pegRNA and the mechanism of the PE3 system. (B) Structure of HBB and the locations of HBB mutations. The blue areas denote exons. The numbers indicate the amino acid residues encoded by the three exons. (C) PegRNA sequences (RTT plus PBS) for each mutation. The desired edits are shown in red. (D) Construction workflow of HEK293T cell lines with heterozygous and homozygous mutations.

To demonstrate the editing capabilities of the PE3 system in HBB, we selected mutations including SNVs (−32 (C>A), CD 17 (A>T)), insertions (CD 14/15 (+G), CD 71–72 (+A)), and deletions (CD 31 (−C), CD 41/42 (−TTCT)). The locations of these mutations in HBB are shown in Figure 1B. For each site, we designed three pegRNAs to induce the desired HBB mutations, and successful editable pegRNA (RTT plus PBS) are presented (Figure 1C). To establish the stable HEK293T cells with HBB mutations, HEK293T cells were edited by transfecting plasmids containing the machineries of PE3 systems, followed by selection in culture medium supplemented with puromycin (Figure 1D). Correctly edited cells were verified by sequencing. The cells with heterozygous and homozygous HBB mutations were further cultured for at least 10 weeks. Finally, 12 HEK293T cell lines with various HBB mutations were obtained, and these cells were passaged and preserved accordingly.

Validation of HBB mutations in HEK293T cell lines

To verify the reliability of the genomic DNA sequences in these long-term cultured cell lines, the HBB region of both homozygous and heterozygous mutant cells was amplified for Sanger sequencing. Compared with their respective wild-type counterparts, cells with homozygous mutations had correct sequence variations, while cells with heterozygous mutations had both mutant and wild-type alleles (Figure 2). These results demonstrated accurate editing of HBB mutations, including SNVs, insertions, and deletions, at the desired sites, with no other mutation sites generated. The accuracy of the edits remained stable after 10 weeks of culturing.

Figure 2:

Sanger sequencing verification of established HEK293T cell lines. Genomic DNA samples were extracted from HEK293T cells with wild-type, homozygous mutant or heterozygous mutant HBB alleles. The sense-strand sequencing results at six mutation sites in the HBB gene are shown. The specific HBB mutation for each cell line is listed on the left side. SNVs or insertions are indicated by underlines, while deletions are denoted by slashes.

Evaluation of HBB mutant HEK293T cell lines as reference materials

To validate the suitability of these novel cell lines as reference materials for β-thalassemia genetic testing, we conducted RDB assays (THA-RDB and βTHA-RDB kits) and a melting curve assay (βTHA-Melt kit) with genomic DNA extracted from cell lines with wild-type, heterozygous mutant and homozygous mutant HBB. The results from the THA-RDB and βTHA-RDB kits (Figure 3) showed that all the normal control dots in the wild-type samples were colored, while no mutant dots were observed. In the heterozygous and homozygous mutant samples, indicative mutant dots were observed on the chips, while no other mutant dots were detected. Furthermore, in the βTHA-RDB kits, the normal control dot for each mutation (−32 (C>A), CD 17 (A>T), CD 71–72 (+A), CD 31 (−C), or CD 41/42 (−TTCT)) was colored in heterozygous samples but disappeared in homozygous samples. Similar results, except for those for CD 31 (−C), were observed for the THA-RDB kits. These findings showed that the selected HBB mutations and the heterozygous and homozygous statuses of our novel cell lines were effectively distinguished in RDB assays.

Figure 3:

Reverse dot-blot assay results for established HEK293T cell lines with various HBB mutations. Genomic DNA samples extracted from HEK293T cells with wild type, homozygous mutant or heterozygous mutant HBB were analyzed using two commercial kits, named βTHA-RDB and THA-RDB. The specific HBB mutation for each cell line is listed on the left side. Strips after hybridization and color development are displayed. The blue dots represent the respective normal controls or mutations listed on each strip. The positions of the mutant genotype are indicated by solid red boxes, while those of the representative wild-type genotype are marked by red dashed boxes.

Further validation of these cell lines was conducted through a melting curve assay using the βTHA-Melt kit (Figure 4). Compared to the Tm value of the wild-type genotype, shifts in Tm values were accurately detected in homozygous mutations of −32 (C>A), CD 17 (A>T), CD 71–72 (+A), CD 14/15 (+G), and CD 41/42 (−TTCT). The melting curve analysis of heterozygous mutations revealed Tm values corresponding to both wild-type and mutant HBB simultaneously. These results affirm that the HBB heterozygous and homozygous mutations were accurately introduced into the HEK293T cells, facilitating genotyping via RDB or melting curve analysis.

Figure 4:

Melting curve assay results for established HEK293T cell lines with various HBB mutations. Genomic DNA samples extracted from HEK293T cells with wild type, homozygous mutant or heterozygous mutant HBB were analyzed using the βTHA-Melt kit. The melting curves and corresponding Tm values for the mutant detection channels are presented. The Tm values of wild-type and mutant HBB genotypes are indicated by solid boxes and dashed boxes, respectively. The specific HBB mutation for each cell line is listed on the left side.

Discussion

β-Thalassaemia is one of the most common inherited blood disorders and is recognized as a notable public health concern in Asia, including China [23], 24]. The objective of this study was to develop a set of publicly accessible genomic DNA reference materials from stable cell lines for β-thalassaemia genetic testing. We successfully established 12 genetic stable HEK293T cell lines with HBB mutations using PE3 systems.

Different types of reference materials, such as residual patient samples, plasmids [25], and cell lines [12], 26], are utilized for β-thalassaemia and other heritable disorders. Each type has its own advantages and limitations. Cell lines, unlike plasmid samples, which are prone to contamination due to their high copy number, offer the advantage of mimicking both heterozygous and homozygous genotypes, as well as real sample extraction and testing processes [27]. Compared with lymphoblastoid cell lines (LCLs) immortalized from blood samples collected from mutation carriers, genetically modified cell lines are particularly suitable for β-thalassemia reference materials. Due to the extensive number of HBB mutations and the low frequency of most mutations [4], 28], it is challenging to collect clinical patient samples or establish LCLs from them to obtain reference materials with a wide range of mutations. This is evident in the fact that from China’s National Institutes for Food and Drug Control or Coriell Cell Repositories, only 10–18 types of β-thalassemia variations’ genomic DNA samples from LCLs can be obtained [28]. Moreover, some β-thalassemia testing products require both heterozygous and homozygous reference materials, which can be challenging to obtain due to the low frequency of homozygous carriers [29], 30]. This challenge is exacerbated by the fact that most homozygous carriers of the β+ phenotype are β-thalassemia major, with some cases even resulting in fatality [31]. For these reasons, we chose HEK293T cells to obtain homozygous and heterozygous cell lines with specific mutations through genetically modified methods. The mutations we selected included −32 (C>A), which is a mutation in the gene promoter that corresponds to the β+ phenotype. The other mutations result in the β0 phenotype: CD17 (A>T) leads to nonsense codons, while CD 14/15 (+G), CD 71–72 (+A), CD 31 (−C), and CD 41/42 (−TTCT) cause frameshift mutations [5], 32]. Mutations such as −32 (C>A), CD 14/15 (+G), CD 27/28 (+C), and CD 31 (−C) are considered rare in China, with a prevalence potentially lower than 0.05 % [28]. Our novel cell lines can encompass rare mutations and obtain clinically challenging homozygous genotypes, thereby addressing the limitations associated with plasmids and LCLs as β-thalassemia reference materials.

Gene editing technology, especially CRISPR-Cas systems, provides powerful tools for targeted manipulation of the genome in cells and animals [16]. PE systems are novel CRISPR-based genomic editing methods. As illustrated in Figure 1A, PE systems function independently of DSBs, enabling targeted gene correction rather than gene disruption [33]. The PE3 system has been demonstrated to achieve all 12 single-nucleotide substitutions, small insertions, small deletions and deletions up to 80 bp in HEK293T cells, with an editing efficiency of up to 20 % and avoiding excess indel formation [14]. Thus, the wide-ranging editing capabilities of PE are ideally suited for the diverse mutation types found in HBB. Our study successfully targeted and achieved three typical HBB mutations (SNVs, small insertions, and deletions). Notably, the successful edits of −32 (C>A) and CD 17 (A>T) also showed the expanded editing capabilities of PE compared to those of base editors, which are limited to installing the A to G or C to T transition [34], 35]. Moreover, in the PE system, the desired edits are encoded by the RTT within the pegRNA, minimizing off-target edits compared to CRISPR-Cas9 or base editing [14], 36]. Given the existence of potentially hundreds of β-thalassemia-related mutations in HBB, PE systems with minimal off-target effects provide an optimal strategy for generating mutations in HBB. The use of PE3 systems to generate HBB mutant cell lines in our study also paves the way for using subsequent versions of PEs, such as PE4, PE5 or twinPE [37], 38], to target more gene mutations in α- and β-thalassemia.

For genetic testing applications, reference materials based on cell lines with stable long-term cultivation characteristics and well-defined qualitative and quantitative properties are necessary [11]. Following initial sequencing verification, the cells were cultured for at least 10 weeks and characterized using Sanger sequencing, RDB analysis, or melting curve analysis in this study. The results of DNA sequencing (Figure 2) showed that our cell lines carrying HBB mutations did not exhibit nontarget mutations even after extended culture. Additionally, thalassemia diagnosis in the laboratory typically involves molecular analysis, with RDB and melting curve assays being widely used [6], 39]. The RDB kits used in this study were approved by the China National Medical Products Administration for detecting β-thalassemia mutations. The melting curve kit is also well-developed for β-thalassemia genotyping. Our novel HEK293T cell lines were successfully characterized using RDB and melting curve assays, demonstrating their suitability for quality assurance of β-thalassemia genotyping. It should be noted that CD14/15 (+G) and CD31 (−C), which are rare mutations in clinical practice [29], could not be distinguished for homozygous/heterozygous status using βTHA-RDB or THA-RDB kits. CD31 (−C) was also undetectable with the βTHA-Melt kit. These facts also highlight the current need for reference materials for rare mutations.

Various molecular testing methods, including quantitative polymerase chain reaction (qPCR), digital PCR, and next-generation sequencing (NGS), are also employed for β-thalassemia diagnosis [40]. These innovative technological platforms expand the detection range of HBB mutations causing β-thalassemia disease, increasing the demand for relevant reference materials. It is promising that our cell lines could serve as reliable reference materials for other molecular analysis approaches beyond RDB and melting curve assays.

In conclusion, we used PE3 systems to generate stable HEK293T cell lines with homozygous or heterozygous HBB mutations, providing valuable reference materials for genetic testing of β-thalassemia. Given the ease of manipulation, cost-effectiveness, and rapid proliferation of HEK293T cell lines, these cell lines, along with future ones that include rare mutations, can contribute to a comprehensive set of reference materials for clinically relevant HBB mutations.