Aplicarea tehnologiei CRISPR-Cas9 în tratamentul leucemiei limfocitare cronice cu mutații ale genei P53

1. Introduction

The diagnosis, clinical staging and therapeutic response evaluation of chronic lymphocytic leukemia (CLL) are based on the criteria recommended by the International Workshop on CLL⁽¹⁾.The patients underwent complete physical examinations for the diagnosis of B-cell CLL, presenting with symptoms such as persistent cough, night sweats and retrosternal pain. In CLL, flow cytometry has confirmed that the presence of a clonal B-cell population expressing cluster of differentiation (CD) 5+, CD19+, CD20+/- and CD23+ is usually sufficient to establish the diagnosis⁽²⁾.

The CD38 receptor is considered positive if lymphocytes display stronger staining intensity than granulocytes in the sample, and its expression is also associated with z-chain-associated protein kinase 70, an adverse prognostic marker⁽³⁾. Giemsa staining of these cells revealed atypical morphologies, including irregular cytoplasm, degenerative vacuoles and frequent multinucleation, suggesting profound mitotic defects (Figure 1).

TP53 gene mutations are common in human neoplasia. A single allele mutation can cause hereditary cancer susceptibility syndromes such as Li-Fraumeni syndrome. Variants of this gene encode distinct p53 isoforms that may disrupt transcriptional activity. Notably, the mitotic index was significantly lower in CLL cells harboring biallelic ataxia-telangiectasia mutated (ATM) and TP53 loss than in CLL cells without such genetic alterations.

Approximately 80% of patients with 17p13 chromosomal deletions harbor mutations in the remaining TP53 allele, resulting in the loss of p53 protein function. Non-deletion mutations occur in approximately 4-5% of cases. Although TP53 mutations are generally associated with poor prognosis, not all mutations predict equivalent disruption of the p53 pathway. Moreover, a subset of patients with the 17p13 deletion exhibit an indolent clinical course, suggesting that p53 function can sometimes be preserved⁽³⁾.

TP53 mutations are considered “multi-hit” if one of the following criteria is met: (i) the presence of two distinct TP53 mutations; (ii) a single TP53 mutation with a variant allele frequency >50% or; (iii) a single TP53 mutation accompanied by 17p deletion on karyotyping. Multi-hit TP53 mutations correlate strongly with high-risk disease features, complex karyotypes and poor survival outcomes⁽⁴⁾.

One of the most widely used prognostic scores for CLL is the International Prognostic Index. This score integrates five independent prognostic factors: TP53 deletion and/or mutation (collectively referred to as TP53 dysfunction), immunoglobulin heavy chain variable region (IGHV) mutational status, serum b2-microglobulin, clinical stage and patient age. Elevated levels of b2-microglobulin (≥5 mg/L) and lactate dehydrogenase (>250 U/L) are used to stratify patients into three prognostic risk groups, with corresponding three-year survival rates of 63%, 83% and 93%, respectively. Richter transformation occurs in 17% of patients in the high-risk group, but in none of the patients in the low-risk group⁽⁵⁾.

Identification of isoform p53 protein using ELISA technique

The diagnosis of CLL was supported by cytological examination of peripheral blood smears under micro­scopy, showing an absolute lymphocyte count higher than 5000/µL with less than 10% prolymphocytes in the differential blood count.  In recent years, it has been discovered that the production of certain isoforms of the p53 protein, which exhibit increased stability in B lymphocytes, contributes to carcinogenesis. This finding has facilitated the identification of p53 protein isoforms using different methods, including immunohistochemistry and enzyme-linked immunosorbent assay (ELISA; www.jacksonimmuno.com). Among these, enzyme-linked immunosorbent assay is widely used as an initial screening method for detecting p53 isoforms generated by mutant TP53 genes.

The participants demographics under study and the results of this work will be addressed in a robust and generaliza­ble manner. In the first step of this research, it is recom­mended to be chosen patients in the 55-65-year-old group.

Additionally, patients must be assessed using ultrasound and positron emission computed tomography imaging, which can reveal lymphadenopathy or splenomegaly, including an enlarged spleen characteristic of hematologic diseases.

Principle of the enzyme-linked immunosorbent assay

This assay is based on the principle of sandwich ELISA. Each well of the microtiter plate is pre-coated with a specific capture antibody. When standards or samples are added, the target antigen – in this case, the p53 protein – binds to the capture antibody. In this study, a specific kit for human p53 protein (aa20-25) was used, employing a purified monoclonal antibody (clone DO-1, isotype IgG2a; www.antibodies-online.com). The antibody is suitable for the techniques ICC/IF and ELISA. Species reactivity is for human in conformity with the prospect from manual, Catalog No. LS-F174, as confirmed by Ray Biotech Life, Inc (https://www.raybiotech.com). The PAb 240 antibody was also utilized for its specific binding to the denatured p53 protein. Compatible sample types included both plasma and serum, which were processed using 96-well microplates.

Applications: human-target specific kit. This information is derived from tests reported in peer-reviewed publications or personal communications from the original suppliers. The general protocol recommendations are available on: www.bio-rad-antibodies.com/protocols. This monoclonal p53 antibody recognizes both mutant and wild-type forms of p53 under denaturing conditions (info@raybiotech.com).

The PAb 240 clone recognizes an epitope that is structurally hidden in the wild-type conformation of p53 but becomes exposed after denaturation or in mutant conformations of p53, in which point mutations in the TP53 gene alter the protein structure⁽⁶⁾.

Species reactivity: the antibody reacts with p53 protein from mouse, rat, cow, dog, human, monkey, Chinese hamster and Syrian hamster. Immunogen: gel-purified p53-b-galactosidase fusion protein containing murine p53 from aa 14-389, derived from cDNA clone pSV53C (https://www.novusbio.com). 

Enzyme-linked immunosorbent assay protocol for detecting p53 isoforms in chronic lymphocytic leukemia samples

The reagents, samples and standards are prepared according to the manufacturer’s instructions. Human samples are used as described in the product leaflet section. The sample types included cell culture supernatants, plasma and serum, and assays were performed using 96-well microplates.

A volume of 100 µL of the standard or sample was then added to each well. For blood samples collected with ethylenediaminetetraacetic acid as an anticoagulant, lymphocytes were isolated and prepared for the assay. Plasma was collected using ethylenediaminetetraacetic acid or heparin-coated vacutainers and centrifuged at 4500 rpm (approximately 280×g) for 15 minutes. Following centrifugation, plasma was separated from red blood cells and fractionated into four distinct layers, with lymphocytes forming a visible “buffy coat”. Using a pipette, 100 µL of lymphocyte layer were carefully extracted (Figure 2).

2. Results

2.1. Results in a short preliminary study using human p53 ELISA kit

After analyzing the 85 LLC samples, in different sta­ges of disease evolution, starting with stage zero (stay and watch) and up to stage IV, 20 patients were selected, eligible for this study, to be investigated for the detection of p53 protein isoforms responsible for resistance to oncological treatments of the disease, using rituximab, cyclophosphamide, doxorubicin hydrochloride, vincristine sulfate (Oncovin®) and prednisone, R-CHOP, after two cycles of relapses, representing a group of 16 men and four women, aged 39-85 years old.

Male results. Protein concentration in p-53/µg/dL: 20, 15, 18, 40, 10, 12, 14, 60, 30, 10, 13, 15, 5, 10, 15, 12. Women’s results. Protein concentration in p-53/µg/dL: 140, 30, 13, 10. Normal values of normal cell lines on equipment: ELISA = 10 µg/dL, or 2.5-5 ng/mL. Very high pathological values in the three cases of p53 were calculated in two men, with the value of 60 µg/dL, respectively at 40 µg/dL, and in the case of females, it was calculated in the amount of 140 µg/dL, the frequency of chronic lymphocytic leukemia with transformation into diffuse large lymphoma⁽³⁷⁾.

2.2. Results of CRISPR-Cas9 technology in possible application in treatment of CLL with mutant P53 gene

Clustered regularly interspaced short palindromic repeats (CRISPR) associated protein (Cas)-9 technology can be delivered in several forms: plasmid DNA encoding both Cas9 protein and single-guide RNA (sgRNA), CRISPR mRNA with sgRNA, or as a ribonucleoprotein (RNP), complex consisting of Cas9 protein bound to sgRNA (http://crispr.mit.edu/). Alternative nucleases include smaller Cas9 variants or Cpf1 (Cas12a), which has gained popularity because of efficient transport and distinct protospacer adjacent motif (PAM) requirements, in an alternative approach⁽⁷⁾.  

The CRISPR-Cas9 system is a versatile tool for introducing targeted mutations or insertions into the genomic DNA. The system comprises a short non-coding guide RNA (gRNA) with two components: a CRISPR RNA (crRNA), which provides sequence specificity, and a trans-activating crRNA (tracrRNA), which binds Cas9. SpCas9 is a colossal CRISPR nuclease, with a size of 1368 amino acids and a moderately relaxed protospacer adjacent motif (PAM; 5¢-NGG) compared to its orthologs⁽⁸⁾. CRISPR in the functional genomics of hematological malignancies gRNA directs Cas9 to the target genomic sequence, where Cas9 introduces a double-strand break (DSB). The sgRNAs were designed using the online CRISPR design tool (http://crispr.mit.edu/) to target TP53 exons – Scheme 1.

Following DNA cleavage, repair is mediated by endogenous cell’s pathways. Non-homologous end-joining often results in indels that can disrupt gene function, whereas homology-directed repair enables precise insertion or correction if a DNA repair template is provided. Despite these improvements, homology-directed repair efficiency remains low in non-dividing cells, limiting therapeutic integration. Moreover, DSBs can lead to deleterious outcomes, such as large deletions or chromosomal translocations⁽⁷⁾.

In normal fluorescence in situ hybridization analysis, two red signals (for the TP53 locus) and two green signals (for the ATM locus) are observed. In CLL, genetic heterogeneity of susceptibility has been mapped to the most distal band on chromosome 17p13 (Figures 3 and 4).

Caspase activation plays an important role downstream of CRISPR-induced apoptosis. Caspases cleave cytoskeletal proteins, nuclear pore components, the nuclear lamina, DNAase-inhibitory proteins and poly-adenosine diphosphate-ribose-polymerase, ultimately degrading chromatin and nuclear structures. Mitochondria contribute by activating procaspase-9 through adaptor proteins (https://eu.idtdna.com/site/order/designtool/index/CRISPR_CUSTOM)⁽⁸⁾.

Loading large DNA payloads into CRISPR systems

At the most basic level, CRISPR requires two components: a Cas nuclease and a gRNA. Together, they form an RNP complex that targets a 20-base protospacer adjacent to a PAM sequences, which varies depending on the Cas enzyme used. Once bound, Cas9 generates blunt or staggered DSB. The gRNA spacer sequence was complementary to the protospacer, ensuring target Negative Controls (Lenti Array CRISPR Negative Control Lentivirus, Non-targeted Human, 1x10⁷ TU/mL) (www.neb.com/Genome Editing).

Negative controls are non-targeting gRNA sequen­ces that do not recognize any sequence in the human genome.Negative controls are available in multiple pack sizes. Negative controls are used during assay development and as plate controls when running screenings.

Positive controls – True Guide crRNA Positive Control, AAVS1 (Human) 2 nmol A35515

Positive controls are validated gRNA sequences that have demonstrated high-level editing efficiency in various cell types, with editing efficiency up to 90% in some cell types. Individual Lenti Array and True Guide gRNAs against specific genes are available. Positive controls are used during assay development to determine conditions that provide maximum editing efficiency in cell models (http://crispr.mit.edu/).

Recent advances in prime editing have enabled the insertion of larger DNA sequences without DSBs. GRAND editing, as described by Zhang et al., 2023⁽¹³⁾, uses pairs of prime editing gRNAs to insert up to approximately 1 kb of DNA. However, the efficiency drops markedly when the insertion length exceeds approximately 400 bp. Alternative approaches are being developed, such as preprint methods that uses dual-prime editing gRNAs to improve long-fragment integration⁽¹⁰⁾.

The Lenti-Array library product line offers the flexibility for expanding CRISPR-Cas9 screening capabilities, and can help make the next big discoveries (Lenti-Array sgRNA lentiviral, 1x10⁶ TU/mL 200 µL A32042). Donor DNA design and synthesis: Invitrogen™ Gene-Art™ gene synthesis services offer chemical synthesis and sequence verification of virtually any desired genetic sequence, making them ideal for producing donor DNA (www. thermofisher.com/gene synthesis).

3. Discussion

Analysis of isoform and mutant P53 gene

A total of 12 human p53 protein isoforms are identified: p53a, p53b, p53g, ∆40p53a, ∆40p53b, ∆40p53g, ∆133p53a, ∆133p53b, ∆133p53g, ∆160p53a, ∆160p53b and ∆160p53g. These isoforms are expressed in a tissue-dependent manner importantly; p53a is not expressed alone (https://hgvs-nomenclature.org/). Missense mutations affecting direct contact between p53 protein and DNA can be retrieved from the International Agency for Research on Cancer database (www.iarc.who.int), specifically codons 239, 241, 248, 273, 275, 277 and 280. The official nomenclature for describing sequence variants is described by the Human Genome Variation Society (HGVS) (http://www.hgvs.org/mutnomen/) – Scheme 2.

In a previous study, the TP53 gene chip was tested in collaboration with a partner from the International Agency for Research on Cancer (www.iarc.who.int). Missense mutations in structural p53 DNA-binding motifs were localized to the L2 and L3 loops, which interact with DNA in the minor groove (codons 164-194 and 237-250, respectively), or within the loop-sheet-helix motif, which interacts with DNA in the major groove (codons 119-135 and 272-287). These DNA-binding motifs are part of the DNA-binding domain (DBD; codons 102-292)⁽¹¹⁾.

All mutations included in the TP53 database were carefully analyzed and manually reviewed using Mutalyzer (https://mutalyzer.nl/). In solid tumors, TP53 is mutated or deleted in approximately 50% of the cases. In contrast, TP53 aberrations are rare in leukemia, ranging from 5% to 10% at the time of diagnosis (http://www.hgvs.org/mutnomen/). 

Notably, hematopoietic stem cells (HSCs) carrying TP53 missense mutations in the DBD demonstrate a competitive fitness advantage HSCs harboring monoallelic TP53 inactivation⁽¹²⁾. Although TP53-mutant HSCs may promote self-renewal, they do not induce overt leukemic transformation, indicating that the presence of mutant TP53 alone is insufficient to initiate leukemia. Additional selective pressures are required for clonal expansion, evolution and transformation⁽¹³⁾. Importantly, TP53 mutations are often missed during diagnosis using routine fluorescence in situ hybridization screening⁽¹⁴⁾.

The ELISA test should be followed by a test to verify the deletion status of the P53 gene after the diagnosis of B-CLL. According to the results of an interphase FISH test with the Abbott Molecular (Vysis), ATM/TP53 probe are presented in the literature in approximately 80% of all patients with CLL who have at least one of the four common chromosomal changes: a deletion in chromosome 13q14.3 (del(13q)), del(11q), del(17p), or trisomy 12. The normal signal pattern is represented by the presence of two specific signals labeled with red fluorochrome (TP53 locus) and two specific signals labeled with green fluorochrome (ATM locus).

Genetic heterogeneity is susceptibility for chronic lymphocytic leukemia. Susceptibility loci have been mapped to the most distal band on the short arm of this chromosome (17p13).

Advanced diagnostic tools

For the confirmatory diagnosis of CLL cases with p53 isoforms expressed in the nucleus and cytoplasm, next-generation sequencing (NGS) is essential. NGS can detect the full spectrum of mutations, leading either to mutant p53 protein expression (e.g., missense mutations) or to the complete loss of p53 expression (e.g., nonsense mutations, frameshifts, or splice-site alterations). This method provides a high sensitivity across multiple sample types (xGen NGS Solutions Builder Tool).

Modern molecular biology and bioinformatics tools, such as polymerase chain reaction, NGS and karyotyping, are indispensable for understanding the pathogenesis of hematologic malignancies. Pan-cancer NGS enables the detection of TP53 variants, including single-nucleotide variants, monoallelic or biallelic copy number variants, insertions/deletions (indels), translocations and gene fusions.

Missense mutations typically result in aberrant p53 protein expression with a dominant-negative effect, whereas null mutations (e.g., nonsense, splice-site and frameshift mutations) lead to a complete loss of protein expression. Both null and missense TP53 mutations had similar effects on survival outcomes (NGS-SNP, RRID:SCR_005182; http://stothard.afns.ualberta.ca/downloads/NGS-SNP/). Therefore, TP53 sequencing using NGS is recommended before treatment initiation in all patients enrolled in clinical trials, as outlined in the National Comprehensive Cancer Network Guidelines⁽¹⁵⁾.

Genomic DNA is often isolated from the bone marrow using formalin-fixed, paraffin-embedded samples, which are the most commonly available materials for molecular testing. DNA extracted from lymphocytes in formalin-fixed, paraffin-embedded samples is usually fragmented (approximately 180 base pairs [bp]) and requires ≤20 ng of DNA input. Sensitivity can reach a 1% variant allele frequency by combining short-amplicon polymerase chain reaction with variant-enriched single-base extension chemistry, which is more sensitive than most conventional NGS assays⁽¹⁶⁾. All analyses will be performed with the written agreement of the patients, for the experimenters performing surgery, anatomical measures and data analysis, the genotypes and treatments, in order to obtain correct results.

TP53 mutation landscape

Most TP53 mutations are missense variants, although nonsense, splice-site, and frameshift mutations also occur, with the majority clustered within the DBD (exons 5-8). The six common mutation hotspots included R175H, Y220C, M237I, R248Q, R273H and R282W. These mutations vary in their impact, ranging from complete loss of tumor suppressor function to partial loss or even gain-of-function activity⁽¹⁷⁾.

Optimizing the reverse transfection of synthetic gRNAs into Cas9-expressing cell lines involves strict controls, continuous monitoring and fine-tuning of delivery conditions. CRISPR screening strategies achieve stable gene editing through plasmid DNA integration, while minimizing innate immune responses⁽¹⁸⁾.

Key components of CRISPR-Cas9 technology

Key components of the CRISPR-Cas9 technology include:

(i) gRNA: laboratory-designed RNA that locates the target gene. Chimeric sgRNAs direct Cas9 to create DSBs near the PAM sequences. Synthetic two-part gRNAs (separate crRNAs and tracrRNAs) were used and HDR-Enhancer.

(ii) TracrRNA: partners with crRNA to form a two-RNA structure that directs Cas9 to induce DSBs at the target site of CRISPR Gene Editing, Alt-R CRISPR gRNA Libraries.

(iii) Positive and negative controls: CRISPR experiments employed. TrueGuide crRNA-positive controls and appropriate negative controls to validate editing efficiency and minimize off-target effects⁽¹⁹⁾.

Future perspectives: CRISPR in chronic lymphocytic leukemia

The potential application of CRISPR technology in CLL follows models established in other hematologic disorders, such as b-thalassemia and sickle cell anemia – severe hereditary hemoglobinopathies, where CRISPR-Cas9 has been successfully used for gene replacement and correction. These advances highlight CRISPR as a promising tool for targeted gene therapy of CLL. CRISPR systems have also been used during assay development and as plate controls in screening experiments^(20-22).

Assessing gene editing efficiency

Following transfection with the CRISPR-Cas9 system, it is essential to evaluate the gene editing efficiency by monitoring cleavage at control loci. The conditions with the highest efficiency were selected for the subsequent experiments (https://www.cbinsights.com/research/what-is-crispr/?utm_source=CB+Insights+Newsletter&utm_campaign=32e18eb638-Top_Research_Briefs_02_09_2019&utm_medium=email&utm_term=0_9dc0513989-32e18eb638-90182665).

The GeneArt Genomic Cleavage Detection Kit provides a rapid and reliable method for measuring CRISPR-Cas9 cleavage efficiency in pooled cell populations⁽²³⁾. Nonviral delivery platforms, such as lipid nanoparticles, cell-penetrating peptides, DNA “nano-claws” and gold nanoparticles, have been explored; however, efficient delivery remains a significant challenge in hematological malignancies⁽²³⁾ (https://www.cbinsights.com/research/what-is-crispr/?utm_source=CB+Insights+Newsletter&utm_campaign=32e18eb638-Top_Research_Briefs_02_09_2019&utm_medium=email&utm_term=0_9dc0513989-32e18eb638-9018266).

Current approaches allow CRISPR-Cas9-mediated repair or replacement of mutant TP53 genes at endogenous promoters; however, they require prolonged cell culture and selection of corrected HSCs. Further progress in HSC growth and expansion is required before clinical application⁽²⁴⁾. The detailed methodology for treating CLL using CRISPR-Cas9 technology targeting mutant TP53 is described at: https://eu.idtdna.com/site/order/designtool/index/CRISPR_CUSTOM.

a) Tissue culture

Induced pluripotent stem (iPS) cells are cultured in Essential 8™ medium on vitronectin-coated plates under serum-free conditions of the manufacturer’s recommendations. Cells are passaged using ReLeSR and supplemented with Revita Cell. The medium is replaced every 24 hours (https://promocell.com/de_de/isolation-of-patient-derived-primary-cancer-cells.html). Select, pure, high-quality growth factors to help you achieve consistent cell culture (thermofisher.com/growth factors). Individual requirements: thermofisher.com/custom media or thermofisher.com/cell culture plastics (Scheme 3).

b) Media, supplements and reagents

Gibco cells culture media are used to maintaining mammalian cells, ensuring reproducibility and consistency of the experimental results (www.thermofisher.com/media)⁽²⁵⁾.

c) Transfection

Transfection are performed using the Lipofectamine™ reagents and Neon™ electroporation. Cleavage efficiency is assessed 72 hours post-transfection with the Gene Art Genomic Cleavage Detection Kit (www.thermofisher.com/media)⁽²⁶⁾.

Hematopoietic stem cells are isolated from CLL of patients, and Cas9 with sgRNAs are delivered as RNP complexes or plasmids. Successfully edited HSC are evaluated and reintroduced into conditioned patients. The control of edited P53 gene is verified with pKi67 protein index. Clinically, the index of proliferative studies of different types of cancer pKi67 protein index has been shown to be higher in malignant tissues with poorly differentiated tumor cells compared to normal tissues. A past cutoff of 20% was established for classifying cancer tumors as highly proliferative.

The Ki67 protein has a half-life of approximately 1-1.5 hours. It is present during all active phases of the cell cycle (G1, S, G2 and M), but it is absent in resting cells (G0). In the later phases of mitosis (during anaphase and telophase), Ki67 levels decrease sharply. Studies on Ki67 have indicated that it undergoes proteasome-mediated degradation during the G1 phase and upon cell-cycle exit⁽³⁸⁾. This strategy is particularly suitable for hematologic disorders caused by single-gene mutations affecting myeloid and/or lymphoid lineages.

d) Hematopoietic differentiation

Hematopoietic stem cells are the central drivers of hematopoiesis, producing both cells’ common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs). CLPs give rise to lymphocytes via megakaryocyte-erythroid progenitors and granulocyte-monocyte progenitors⁽²⁷⁾.

B-cell development begins in the bone marrow of pluripotent HSCs. Early progenitors express CD34+, TdT+, CD19+, CD79a+, cyCD22+ and HLA-DR+, but are CD10- and sIgM-. Precursor B cells are characterized by CD34+, TdT+, CD45^dim+, CD19+, CD79a+, cyCD22+, HLA-DR+, CD10^int+, cyIgM+ and sIgM+. As B cells mature, they progress from immature CD10+ to naive, and finally to mature CD10-B cells, ultimately populating both bone marrow and peripheral blood (Figure 5)⁽²⁸⁾ – Scheme 4.

e) Allogeneic hematopoietic cell transplantation

Currently, allogeneic hematopoietic cell transplantation remains the only potentially curative option for TP53-mutated CLL. HSCs appeared as immature CD34+/CD38+cells. Immunophenotypic testing should also evaluate CD45 isoforms (e.g., CD45RB, CD45RO and CD45RA) in CD34+ cells to distinguish malignant HSCs from CLPs. Ex vivo HSC properties can be assessed by culturing cells in semi-solid media and quantifying their ability to form multi-lineage colony-forming units (Human CRISPR Library v.1.1. Type: CRISPRn. Format: Pool Enzyme. Druggable CRISPR Library; https://www.addgene.org)⁽²⁹⁾.

4. Materials and method

Sample preparation

For sample preparation, 2 mL of bone marrow aspirate was required, which is sufficient for analysis, and can be stored at 4°C for up to 72 hours. Samples obtained by flow cytometry should be accompanied by an unstained smear of the same sample during transport to the laboratory. For sample processing, wash stain methods are recommended, with washing as the preferred approach. Samples are diluted to obtain 10⁵-10⁶ cells per tube and aliquoted into 25-100 µL volumes. Care must be taken during centrifugation to avoid the destruction lymphoblasts. Labeling is a thermodynamic process between antibodies and antigens, and should be performed for no longer than 15-20 minutes, at room temperature, in the dark. Both positive and negative control fluorescence. Normal residual cells or unstained cells are also included as reference controls.

Routine flow cytometric analysis of bone marrow aspirates enables the identification of lymphocyte progenitors. Proliferated B lymphocytes are characterized by CD45+, CD19+, CD90^dim/partial, CD5+, CD23+, CD200+, CD43+, sIg phenotypes dim. Lymphoid progenitors are defined as CD34+, CD38-, CD90^dim/-, CD45RA+, CD135+ and CD10+ (https://www.flowjo.com/solutions/flowjo; RRID:SCR_008520 corresponds to the Antibody Registry). To perform cell isolation as described above, use the following reagents: EasySep™ HLA Chimerism Whole Blood CD19 Positive Selection Kit (Catalog #17874), EasySep™ HLA Chimerism Whole Blood CD3 Positive Selection Kit (Catalog #17871), EasySep™ HLA Chimerism Whole Blood CD19 Positive Selection Kit (Catalog #17874), EasySep™ HLA Chimerism Whole Blood CD3 Positive Selection Kit (Catalog #17871), Stemcell Technology (https://www.stemcel.com).

Modern cytometers automatically perform compensation using integrated software. Abnormal populations (blasts) are analyzed based on CD45 receptor scatter patterns. Cells within this progenitor reservoir have the potential to generate all lymphocyte subpopulations, macrophages and dendritic cells, and are classified as multilymphoid progenitors⁽³⁰⁾.

Automatic separation of multiple cell types from a single sample

Cell separation for chimerism analysis was performed using an EasySep™ kit. Example of  markers and applications include: hematopoietic progenitor cells (CD34); B progenitors (CD34+, TdT+, CD19+, CD79a+, cyCD22+, HLA-DR+, CD10-, cyIgM-, sIg-); B precursors (CD34+, TdT+, CD45^dim+, CD19+, CD79a+, cyCD22+, HLA-DR+, CD10^int+, cyIgM+, sIg+); and mature B cells (CD5+, CD19+, CD20+/-, CD23+).

Differentiation of hematopoietic stem/progenitor cells into lymphoid progenitors

CD34+ hematopoietic stem/progenitor cells are seeded in untreated tissue plates coated with lymphoid expansion reagents, at protocol-specified cell densities.

Medium changes are performed at regular intervals. By day 14, lymphoid-expanded cells are harvested and reseeded at a density of 5×10⁴ live cells/mL in uncoated tissue culture plates using NK differentiation medium supplemented with 1 mM UM729.

A subset of cells was cryopreserved in CryoStor-CS10, where the remainder was stained with antibodies against CD5, CD7, CD43, CD45 and CD34, to monitor the progressive loss of CD34 expression during differentiation^(31,32).

Cell analysis and instrumentation

Cell counts are performed using the Invitrogen™ Countess™ II FL Automated Cell Counter, which provides bright-field and dual-channel fluorescence analysis to monitor viability, transfection efficiency and protein expression. Red blood cells are lysed using red blood cell lysis buffer, followed by two washes with phosphate-buffered saline. Cells are analyzed on a BD FACS Aria flow cytometer, and data are processed using Flow Jo software to assessing green fluorescent protein (GFP) and red fluorescent protein (RFP) signals to classify edited populations: -P53^MUT: GFP+/RFP-; -P53-del(13p) and ATM: GFP+/RFP+⁽³³⁾.

The CRISPR-Cas9 gene editing involves collecting a patient’s progenitor T cells (a type of immune-system cell) at a treatment site (such as a hospital), confirming these cells by flow cytometry transporting the cells to a manufacturing site, reprogramming the genetic material of the T cells transporting the cells back to the treatment site, and infusing the modified edited cells into the patients. Following collection, the edited stem cells are reinfused into patients after cytoreduction of the bone marrow, with the aim of reducing the hematopoietic burden of mutant TP53 clones (Scheme 5)⁽³⁴⁾.

The Adult Genotype-Tissue Expression (GTEx) project is a comprehensive public resource for the study of tissue-specific gene expression and regulation (GTEx portal: https://gtexportal.org). Samples were collected from 54 non-diseased tissue sites across nearly 1000 individuals, primarily for molecular assays, including WGS, WES and RNA-Seq. All data files generated in the GTEx project are available at references. All raw sequence data files (DNA and RNA), along with the full donor metadata files, will protect access data⁽³⁵⁾.

Time-tested and trusted, the Gibco cell culture media include products designed to support the growth and maintenance of a variety of mammalian cells and cell lines (thermofisher.com/media). Select pure, high-quality growth factors to help achieve consistent cell culture (https://www.thermofisher.com/growth factors). Individual requirements: thermofisher.com/custom media, or thermofisher.com/cell culture plastics⁽³⁶⁾.

5. Conclusions

Clustered regularly interspaced short palindromic repeats-based functional genomics provides a powerful platform for identifying genes most affected by single-gene disruption, assessing gene-gene interactions, and characterizing mutation profiles associated with adverse outcomes in CLL.

The CRISPR-Cas9 gene editing shows significant promise as a therapeutic strategy for TP53-mutant CLL by enabling precise gene correction, functional replacement, or pathway disruption. Continuing improvements in delivery systems, editing efficiency and safety monitoring are critical for clinical translation⁽³⁸⁾.

Our goal was to develop a framework and an implementation support system that would be practically applicable for a broad range of journals in the life sciences. In many ways, the MDAR Framework is a joint iteration on our collective previous experience with guidelines, checklists and editorial requirements, toward a harmonized and practical guideline for minimum reporting requirements.   

Acknowledgments: Not applicable.

Funding: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authors contributions: All authors contributed to the intellectual content of this paper, and in this work of research they have accomplished the following three requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Conceptualization: Aurelian Udriştioiu. Data curation: Ioan-Ovidiu Gheorghe. Formal analysis: Manole Cojocaru. Investigation: Liviu Martin. Methodo­logy: Delia Nica-Badea. Writing original draft: Adrian-Victor Tetileanu. Writing-review and editing: Aurelian Udriştioiu.

Ethics approval and consent to participate: Not applicable.

Availability of data: Data are available from the corresponding author upon reasonable request.

Ethics approval statement: All experiments were performed following relevant guidelines and regulations in the Declarations section. ANMCS: Accreditation. All methods were performed according relevant guidelines and regulations of the Declaration of Helsinki – Ethical Principles for Medical Research Involving Human Subjects.

Consent for publication: Not applicable.

Availability of data availability statement and permission to reproduce material from other sources: Not applicable.

Corresponding author: Aurelian Udriştioiu E-mail: aurelianu2007@yahoo.com

Conflict of interest: none declared.

Financial support: none declared.

This work is permanently accessible online free of charge and published under the CC-BY licence.