CRISPR-Cas9-based Strategies for Acute Lymphoblastic Leukemia Therapy

Abstract

Defeating cancer as leukemia has been an up and down challenge. However, leukemia must be treated from the roots. Nowadays, the CRISPR-Cas9 system provided scientists the ability to manipulate the genetic information to correct mutations, rewrite genetic code, or edit immune cells for immunotherapy purposes. Additionally, such system is used for basic and clinical approaches in leukemia therapy. Lymphoid cancers including acute lymphoblastic leukemia (ALL) can be treated by performing gene editing or enhancing immune system through CART cells. Here, we present and detail therapeutic applications of the CRISPR/Cas9 system for immune cell therapy, and knock-out or knock-in of main genes promoting leukemogenesis or ALL progression. We also described current and future challenges, and optimization for the application of CRISPR/Cas9 system to treat lymphoid malignancies.

1. Introduction

Acute Lymphoblastic Leukemia (ALL) is a type of hematological cancer that affects children and adults around the world. Since the first chemotherapeutic drugs were developed to treat this disease, the survival rate has increased. However, side effects represent one of the main challenges to defeat in ALL. Current therapy for ALL provides selectivity to avoid side effects as much as possible. Still, more specific and effective treatments are necessary. Indeed, gene therapy points to be the most promising future medicine to defeat cancer as ALL. The main objective of gene therapy is to correct mutations that promote leukemogenesis, thereby counteracting or remedying the conditions caused by malfunctioning genes. The most studied and effective technology to alter DNA is the CRISPR-Cas9 system first used to target DNA in 2013. In this chapter, the mechanisms of the CRISPR-Cas9 technology as well as their current and highlight strategies to treat ALL will be explained.

2. Acute lymphoblastic leukemia

2.1 Definition and statistics

Acute lymphoblastic leukemia (ALL) is a hematologic malignancy characterized by impaired differentiation, high proliferation, and accumulation of both T and B lymphoblast in the bone marrow, peripheral blood, and/or extramedullary sites [1, 2]. Children, adolescents, and young adults comprise 70% of ALL cases [3]. The incidence of ALL in the United States (US) is 1.8 per 100,000 for all age groups and 3.5 per 100,000 for ages 0–19. Survival in ALL is strongly influenced by age with five-year relative survival rates of 92.1% in <15 years, 64.1% in 15–39 years, and < 41% in >39 years [4].

2.2 Genetic alterations

2.2.1 B cell ALL

Subtypes of B-ALL are characterized by chromosomal alteration (aneuploidy or chromosomal rearrangements), which promotes deregulation of proteins by chimeric genes formation, and upregulation of genes by juxtaposition with a strong enhancer (Table 1). The risk stratification can be established based on such chromosomal abnormalities. Both copy number alterations and sequence mutations on lymphoid transcription factors contribute to leukemogenesis as secondary genomic events [5, 6].

Category	Frequency	Description	Prognosis
High hyperdiploid (gain ≥5 chromosomes)	Children 25% AYAs and adults 3%	Mutations in RTK-RAS signaling pathway and histone modifiers	Favorable
Near-haploid (24–31 chromosomes)	Children 2% AYAs and adults <1%	Ras-activating mutations Inactivation of IKZF3	Poor
Low-hypodiploid (32–39 chromosomes)	Children <1%; AYAs 5%; Adults >10%	TP53 mutations; Inactivation of IKZF2; Deletions of CDKN2A/B and RB1	Very poor
MLL (KMT2A) rearrangements	Infants >80%; Children <1%; AYAs 4%; Adults 15%	Low number of additional somatic mutations, commonly in kinase PI3K-RAS signaling pathway	Very poor
t(12;21)(q13;q22) translocation encoding ETV6-RUNX1	Children 30%; AYAs and adults <5%		Favorable
t(1;19)(q23;p13) translocation encoding TCF3-PBX1	Children, AYAs, and adults ≈5%	Association with CNS relapse	Favorable
t(1;19)(q23;p13) variant of the TCF3-HLF	<1% ALL		Poor
Philadelphia chromosome, t(9;22)(q34;q11) translocation encoding BCR-ABL1	Children 2–5%; AYAs 6%; Adults >25%	Common deletions of IKZF1, CDKN2A/B, and PAX5	Poor (improved with tyrosine kinase inhibitors)
Philadelphia chromosome -like ALL	Children 10%; AYAs 25–30%; Adults 20%	Rearrangement of CRLF2 (≈50%); Rearrangement of ABL-class tyrosine kinase genes (≈12%); Rearrangement of JAK2 (≈10%); EPO receptor (≈ 3–10%); Mutations activating JAK–STAT (≈ 10%) and Ras (≈2–8%) signaling pathways	Poor
DUX4- and ERG deregulated ALL	5–10% ALL	Distinct gene expression profile; most cases present focal ERG deletions	Favorable, including if coexistence of IKZF1 mutation (≈ 40% patients)
ZNF384-rearranged ALL	Children 5%; AYAs and adults 10%	ZNF384 rearranged with a transcriptional regulator or chromatin modifier (EP300, CREBBP, TAF15, SYNRG, EWSR1, TCF3, ARID1B, BMP2K, and SMARCA2)	Intermediate

Table 1.

Main genetic subtypes of B-cell acute lymphoblastic leukemia.

2.2.2 T cell ALL

T-ALL is the result of a multistep process where accumulation of genetic mutations alters the normal control of cell growth, differentiation, proliferation, and survival during thymopoiesis (Table 2). The genetics of T-ALL is highly heterogeneous, with abnormalities in almost all patients, and it accounts for approximately 15% and 25% of pediatric and adult ALLs, respectively [5, 6].

Category	Frequency	Description	Prognosis
TAL1 deregulation	50% of T-ALL Children<adults	Overexpressed oncogenic transcription factor t(1;7)(p32;q35) and t(1;14)(p32;q11) translocations and interstitial 1p32 deletion	Generally favorable
LMO2 deregulation	50% of T-ALL children	Overexpressed oncogenic transcription factor t(11;14)(p15;q11) translocation and 5´ LMO2 deletion	Generally favorable
TLX1 (HOX11) deregulation	50% of T-ALL Children<adults	Overexpressed oncogenic transcription factor t(10;14)(q24;q11) and t(7;10)(q35;q24) translocations	Good
TLX3 (HOX11L2) deregulation	50% of T-ALL Children and adults	Overexpressed oncogenic transcription factor t(5;14)(q35;q32) translocation; commonly fused to BCL11B, also a target of deletion and/or mutation	Poor
MLL rearrangements	Children	Multiple partners; disruption of HOX gene expression and of self-renewing	Poor
9q34 amplification encoding NUO214-ABL1	8% of T-ALL Children	Amenable to tyrosine kinase inhibitors, also identified in high risk B-ALL; other kinase fusions identified in T-ALL include EML1-ABL1, ETV6-JAK2, and ETV6-ABL1
t(7;9)(q34;q34.3)	Children	Rearrangement of NOTCH1
NOTCH1 mutations	80% of T-ALL; children and adults	Constitutive activation of NOTCH signaling Impairment of differentiation and proliferation	Overall favorable
FBXW7 mutations	Present in 80% of patients; children and adults	Loss of function mutations; Impairment of differentiation and proliferation, usually evaluated in combination with NOTCH1
Early T-cell precursor ALL	Children and adults	Immature immunophenotype; expression of myeloid and/or stem-cell markers; poor outcome; genetically heterogeneous with mutations in hematopoietic regulators, cytokine and Ras signaling, and epigenetic modifiers

Table 2.

Main genetic subtypes of T-cell acute lymphoblastic leukemia.

2.3 Treatments

2.3.1 Chemotherapy

Front-line treatment for ALL consists of a multiagent chemotherapy regimen, typically divided into 3 phases: induction (combination of steroids, anthracyclines, and vincristine), consolidation, and long-term maintenance (over a 2 to 3- year period), and recommend central nervous system (CNS) prophylaxis [5]. Despite high rates of complete remission (CR) (80–90%), about 20% of pediatric and 40% of adult patients will relapse [1, 7]. This treatment can also affect some normal cells in the body, leading to side effects such as hair loss, diarrhea, constipation, and loss of appetite, among others. Besides, affects normal cells in bone marrow leading to leukocytopenia (increased risk of infections), thrombocytopenia (easy bruising or bleeding), and/or anemia (fatigue and shortness of breath) [5, 8].

2.3.2 Immunotherapy

Immunotherapy is the use of medicines to help patient’s immune system to recognize and destroy cancer cells in a more effective way [9]. A broad range of immunotherapy strategies has been developed to overcome the failure of front-line treatment as well for patients with relapsed/refractory (R/R) disease [10], which includes the following:

• Monoclonal antibodies include unconjugated antibodies and antibody-drug conjugate (ADC). Antibody therapy can lead to direct apoptosis, complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated cytotoxicity (ADCC). The targets for ALL include CD19, CD20, CD22, and CD52 [11, 12].

• Bispecific antibodies are single chain variable fragments (Scfv) consisting of at least two different specific antibodies one for tumor-associated surface antigens and the other for surface antigens on effector cells (CD3 ε on T cells) redirecting T cells to lyse malignant cells: CD3/CD19 and CD3/CD20 bispecific T-Cell Engagers (BiTE) [13, 14].

• Chimeric antigen receptor -T (CART) are genetically engineered cell membrane binding receptors, which can activate T cells by linking from the extracellular antigen binding region to the intracellular signal domain via the spacer, whose effect on target antigen is independent of major histocompatibility complex MHC [15].

Even though immunotherapy is the most promising tumor treatment, the advances in molecular biology tools have made it possible to manipulate human DNA by different strategies to treat diseases at the roots. One of the main tools for precise gene editing is CRISPR-Cas9, which allows simple, cost-efficient human cells and another eukaryotic genome editing.

2.4 Overview of CRISPR-Cas9

Clustered regularly interspersed short palindromic repeats (CRISPR), first found in 1987, function as part of an adaptive prokaryotic immune system (CRISPR-associated system, Cas) against phage infection and plasmid transfer in nature [16, 17]. CRISPR-Cas systems are grouped into two classes and subdivided into six types. Class 1 consists of types I, III, and IV, while class two consists of types II, V, and VI. Class 2 systems act by single-subunit effector proteins such as Cas9 and Cas12a and are more precise and efficient for genome engineering than class 1 systems. CRISPR-Cas9 recognizes target DNA through CRISPR RNA (crRNA) and trans activating RNA (tracrRNA), which form a single guide RNA (sgRNA) that activates and guides Cas9 to bind target DNA, which is subsequently cleaved to form a double-strand break (DSB)(Figure 1) [18].

Figure 1.

CRISPR-Cas9 mechanism to disrupt or edit genes precisely.

Since DSB is repaired in a host cell, it may introduce DNA mutations leading to genetic changes when non-homologs end joining (NHEJ) predominates as repair mechanism. NHEJ is an error-prone repair mechanism that often leads to insertions or deletions (indels). These indels can cause frameshift mutations, large deletions or inversions using two adjacent DSBs, genomic rearrangements, NHEJ-mediated homology-independent knock-in, premature stop codons, or/and nonsense mediated decay to the target gene, resulting in a loss-of-function. In contrast, homology-directed repair (HDR), considered a high-fidelity repair, uses assisted recombination of DNA donor templates to reconstruct DSBs. This mechanism can be exploited to introduce well-defined mutations by transferring altered donor templates into targeted cells, which provides the basis to perform precise gene modification, such as gene knock-in/knock-out, insertion, deletion, correction, or replacement [16, 18].

In addition, a nuclease-deficient Cas9 modification (dCas9) can be fused to a variety of effector domains to mediate specific local DNA manipulations as [19]:

• Transcriptional regulation:

  • CRISPR interference (CRISPRi) inhibits transcription by sterically blocking the RNA polymerase [20].

  • CRISPR activation (CRISPRa) binds to the upstream promoter regions and recruits the RNA polymerase, and further activates transcription [20].

• Epigenetic modification:

  • Increasing/repressing DNA methylation of CpG motifs in targeted genomic regions [21, 22, 23, 24].

  • Modifying acetylation of histones to increase the accessibility of genomic regions [25].

• Nucleotide editing: Cas9-nickases (nCas9) were developed to change single DNA nucleotides without introducing DSBs or requiring any homology-directed repair. The system consists of dCas9 coupled to cytidine deaminase domains to induce targeted transitions from C to T or G to A [26, 27, 28].

2.4.1 CRISPR-Cas9-based therapies for acute lymphoblastic leukemia

The next-generation sequencing (NGS) has allowed researchers to analyze genes in a better way and thus, obtain a better correlation between genotype and phenotype. Integrating gene editing to gene sequencing enables researchers to manipulate any described gene or noncoding sequences in a wide variety of organisms including humans. Here, we present and discuss relevant subgroups of genes that were edited as well as therapy strategies based on CRISPR-Cas9.

2.4.1.1 Improving immunotherapy via CRISPR-Cas9

Since immunotherapy provides the most promising clinical outcomes in leukemia, it is attractive to perform the generation of CART cells via CRISPR-Cas9. First, autologous T cells are collected from patients and further genetically engineered to recognize and attack cancer antigens in ex vivo experiments. Cells expressing CART are then transferred into the patient. The use of CRISPR-Cas9 to produce CART cells will allow to treat efficiently different types of cancers and provide higher safety than conventional therapy [29]. Besides, CRISPR-Cas9 can be used to disrupt genes that code signaling molecules or T cell inhibitory receptors to enhance the CART cell’s function.

Immunotherapy can now be improved by using CRISPR-Cas9 to increase the efficacy of CART cells. Such improvement can be performed through knock-out and knock-in mutations in the T-cell receptor α constant (TRAC) locus and CAR against CD19. Previous studies reported that CAR inserted in the AAVS1 locus, which is under control of endogenous regulatory elements, reduces tonic signaling, and blocks differentiation and T cell depletion leading to an increased therapeutic function of CART cells [30]. The disadvantage of CART therapy is due many patients are unable to receive engineered autologous T-cells but can be faced by generating universal infusion products obtained from healthy donors. Nevertheless, the use of “off-the-shelf” CART cell products can promote the induction of graft-versus-host disease (GVHD) because the allogenic T-cells were ex vivo activated causing rejection by the host [31]. To solve this issue, endogenous αβ T-cell receptors (TCRs) are knocked out by CRISPR-Cas9 on transferred donor lymphocytes that interact with/recognize alloantigens, which avoid GVHD. Besides, the deletion or elimination of the subunit of human leukocyte antigen class I (HLA-I) beta-2-microglobulin (β2M) would decrease rapid depletion of allogenic cells expressing foreign HLA molecules [32]. According to preclinical data regarding CRISPR-Cas9-related clinical trials, stimulating NHEJ by cleaving endogenous TCR and β2M through CRISPR-Cas9 will generate universal CD19-directed CART cells to avoid rejection.

The function of CART cells may be negatively regulated by overexpression of negative checkpoint regulators plus up-regulation of cognate inhibitory ligands in the tumor microenvironment. The negative checkpoint regulator programmed cell death 1 (PD-1) as well as other inhibitory receptors such as T-cell Ig and ITIM domain (TIGIT), T cell membrane protein-3 (TIM-3), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and lymphocyte-activation protein-3 (LAG-3) act in a synergistic way to reduce immune cell availability [33, 34]. Currently, the administration of checkpoint inhibitors has been the most effective strategy to reinvigorate such lymphocytes. Unfortunately, severe immune-related side effects affect some patients. In this line, knocking-out multiple genes by CRISPR-Cas9 allows the production of universal, allogenic T-cells lacking multiple negative regulators ready to be transferred into patients [31].

The modification of CART cells against CD19 (CAR19) has been used to effectively treat refractory relapsed B-ALL in infants. The transcription activator-like effectors nucleases (TALENs) technology was used to produce CAR19 and set the basis for engineering T-cells using CRISPR-Cas9 to improve the outcomes [35]. It seems that some type of resistance to CART is observed in 10–20% of treated patients who suffer relapses. These relapses are due to the partial loss of the CD19 epitope [36]. The NGS analysis indicated that frameshift mutations and total loss of exon 2 resulted in alternative splicing of CD19. In this regard, CRISPR-Cas9 was applied to ALL cell lines to eliminate CD19 and to further be reconstituted with different isoforms. It was observed that the depleted isoform of exon 2 was deposited in the cytosol and not in the cell membrane, thus, escaping from CAR19 [37]. The approach of CRISPR-Cas9 was also useful to better understand the mechanisms of immunotherapy resistance.

2.4.1.2 Regulation of transcription factors by CRISPR-Cas9

Transcription factors (TFs) control several cellular processes, and any deregulation could lead to pathogenic mechanisms promoting leukemogenesis. In leukemia, TFs can be amplified, rearranged, deleted, or affected with punctual mutations resulting in gain or loss of function [38]. In consequence, correcting such mutations by CRISPR-Cas9 represents a promising therapeutic strategy in ALL. The TFs PAX5 and IKZF1 are metabolic repressors that limit the amount of ATP available, which decreases cell proliferation rate, but such TFs are altered in about 80% of patients with B-ALL. At this point, CRISPR-Cas9 was used to screen all the possible PAX5 and IKZF1 targets, where NR3C1, TXNIP, and CB2 genes were identified as the effectors of B-cells restriction of glucose and energy supply [38] and proposed as new targets for B-ALL therapy. Also, point mutations on PAX5 and IKZF1 could be corrected by using CRISPR-Cas9 and promoting the HDR mechanism.

On the other hand, TAL1 oncogene is deregulated in T-ALL cells, whose function lies in the regulation of the cell cycle progression. Frequently, deregulation of TAL1 is produced by translocations or deletions, but many patients do not present mutations, which indicates epigenetic-related deregulation [39]. To study the effect of insertion or deletions on the expression of TAL1, CRISPR-Cas9 was used in T-ALL cells. It was observed that methylation acetylation pattern in H3K27 changed in mutant cells [29]. This indicates that mutagenesis and epigenetic modulation cooperate to regulate TAL1 expression. This is an adequate scenario where dCas9 can be used fused with DNA methyl transferases or demethylases, or DNA acethyl or deacetylases to mediate specific local epigenetic modulation. Another potent oncogene deregulated in T-ALL is LMO2, which is a central effector for the formation of large transcriptional complex. It was observed that its overexpression is related to leukemogenesis. Unfortunately, only few LMO2 mutations have been described and its overexpression mechanisms are poorly understood. However, several mutations were introduced into LMO2 by CRISPR-Cas9 identifying those mutations in noncoding regions that promote deregulation in LMO2 expression [40]. In the same manner, such identified mutations can be repaired by CRISPR-Cas9 or epigenetically modulated by dCAS9 leading to normal LMO2 expression.

2.4.1.3 Discovering drug targets by CRISPR-Cas9

Most times, drug failure is related to mutations in genes that code the drug target, but mutations also help to understand the mechanism of action of drugs. Sometimes it is necessary to induce several mutations to study the drug-protein interactions, thus, elucidating their mechanism of action. A notable example is ibrutinib, an inhibitor kinase used to treat ALL where BCR signaling is damaged. The elucidation of its mechanism of action was determined through the generation of different knock-out in ALL cells as follows: Bruton tyrosine kinase (BTK), B lymphocyte kinase (BLK), and BTK/BLK via CRISPR-Cas9. Since overexpression of both BTK and BLK were reported in ALL, the strategy was to eliminate one or both kinases. The BTK/BLK knock-out reduced cell proliferation similar to ibrutinib. However, when ibrutinib was administrated in those ALL cells, the decrease in proliferation was greater suggesting that such drug targets other proteins [41].

In addition, drug specificity and binding site in a protein have been demonstrated by CRISPR-Cas9. Exportin 1 (XPO1) mediates the transport through the nucleus of cell cycle regulatory proteins and tumor suppressor proteins. Such transports are related to development of ALL and poor prognosis. XPO1 inhibitors bind to Cys528 blocking the transport of charged proteins to the cytoplasm. To determine the precise binding site of XPO1 inhibitors as well as its selectivity, a non-synonymous mutation was inserted at Cys528. Such mutation resulted in notorious drug resistance, therefore, demonstrating the high specificity of the inhibitors to XPO1 [38].

Drug efflux-related chemoresistance is the main concern in ALL, where ABC transporter proteins are involved. The proteasome inhibitor carfilzomib (CFZ) significantly improves favorable clinical outcomes for refractory childhood ALL, except in those ALL cells with P-glycoprotein positive t (17;19). By using CRISPR-Cas9, the gene ABCB1 that codes for P-glycoprotein (P-Gp) was knocked-out resulting in sensitization of P-Gp-positive t (17;19) ALL cells to CFZ [42], which is a P-Gp substrate. This finding highlighted the application of CRISPR-Cas9 to combat chemoresistance.

2.4.2 Safety: off-targets

The main side effect of the application of CRISPR-Cas9 is the risk of off-targets, which can knock-out, knock-down, or knock-in other genes permanently. To significantly reduce off-targets, it is recommended to use appropriate sgRNA designing tools, which allow gRNA selection to enhance the high specific DNA manipulation. It has been proposed the prediction of all possible off- target effects for any designed gRNA, at least in clinical trials. Nevertheless, unexpected off-targets have been observed even in off-target free sgRNA [43]. Even though off-target predicting tools (in silico) are not completely precise, such tools must be combined with in vitro assays and NGS techniques to evaluate and identify off-targets and DSBs consequences prior to clinical or preclinical stages. Regarding clinical stages, cell-based genome-wide (CBGW) assays are well recommended to identify cleavage sites under experimental conditions. This is a notorious tool because provides higher safety to patients. To date, CBGW assays such as genome-wide unbiased identification of DSBs enabled by sequencing (GUIDE-seq) as well as linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) are reported to have substantial potential at clinical stages [44].

Since there is not a perfect strategy to detect and avoid off-targets for clinical purposes, current reports indicate that other options available are sgRNA-Cas9 modification. This means the use of Cas9 variants with higher precision or using another type of Cas protein such as Cas12 (Cpf1) in immunotherapy at preclinical stages [45, 46, 47]. Another consideration is the expression of tumor suppressor gene p53 due to its expression in normal cells decreases gene editing by CRISPR-Cas9 system when the HDR mechanism is used. On the other hand, leukemia cells lack p53 function, which makes them easier to edit by CRISPR-Cas9. This is a relevant highlight because, theoretically, most of edited cells would be leukemic cells instead of normal cells [48, 49]. Such statement set the mandatory screening of p53 function in immune cells prior to using CRISPR-Cas9 to produce CART cells.

2.5 Delivery of CRISPR-Cas9 components into target cells

CRISPR-Cas9 components can be introduced as plasmid encoding Cas9 and sgRNA or as Cas9 protein and sgRNA in complex called ribonucleoprotein (RNP). In ex vivo gene editing, microinjection and electroporation have been used successfully to introduce plasmid or RNP. Recently, the transiently mechanical membrane deformation with microfluidic devices provides highly efficient delivery and low cell mortality for ex vivo assays, particularly used to transfect breast cancer and leukemia cells [50]. For preclinical and clinical stages, the transmembrane internalization assisted by membrane filtration (TRIAMF), the induced transduction by osmocytosis, and propane betaine (iTOP) methods are widely and recently used to introduce RNP [51, 52].

Delivering CRISPR-Cas9 components for in vivo applications is more complicated because we must consider the consequences related to off-target mutations in a complete organism as well as immunological responses. The most used method is the use of viral systems to transduce cells. However, such systems have disadvantages such as immune activation by adenoviral vectors and long expression of Cas9 protein by use of lentiviral vectors, which lead to a high frequency of off-targets. The viral systems are being replaced by nonviral or synthetic methods based on the envelop of plasmids or RNP into polymeric, lipid, or inorganic nanoparticles, which are widely used in preclinical and clinical stages [46, 53, 54]. The use of gold nanoparticles has successfully been used to deliver RNP to treat Duchene muscular dystrophy in a mouse model. Also, previous reports indicate that gold nanoparticles are nontoxic, which makes them one of the best options to deliver CRISPR-Cas9 components in RNP format [50].

3. Conclusions

ALL is one of the most common cancers diagnosed in children from 0 to 14 years old. Despite continual improvement in drug development and immunotherapy, it is necessary to focus on the root causes of the disease. For years, scientists dreamt about the possibility to manipulate DNA to correct mutations that promote cancer initiation and progression. Now with the use of CRISPR-Cas9-based gene editing, it is possible to develop effective and specific treatments against ALL. There has been a substantial advance in gene editing technology but still is not possible to ensure 100% specificity to avoid off-target leading side effects. Nevertheless, current research has made it possible to identify the main target genes in ALL and how to edit them by CRISPR-Cas9, mostly through in vitro and ex vivo assays. According to previous reports, CRISPR-Cas9 points to be the most effective tool to develop gene therapy for ALL, but still needs to be optimized to enhance precise gene editing and thus, avoid off-targets.

Conflict of interest

The authors declare no conflict of interest.