Primary immunodeficiencies (PIDs) include more than 400 rare congenital monogenic disorders characterized by impairment of immunity, susceptibility to infectious diseases, autoimmunity, autoinflammatory diseases, allergy and/or malignancy (Tangye et al., 2020). In recent years, there has been an increase in the recognition and diagnosis of previously undefined genetically caused abnormalities in the immune system (Tangye et al., 2020). This has been made possible through the completion of the Human Genome project in the early 2000s, improved definition of clinical phenotypes, and advancement of cost-effective and time-efficient sequencing through implementation of next generation DNA sequencing technologies (Meyts et al., 2016; Bousfiha et al., 2020; Tangye et al., 2020; Gates et al., 2021).

Among these disorders is DOCK8 immunodeficiency syndrome (DIDS) also known as DOCK8 deficiency. Until recently DOCK8 deficiency was termed DOCK8-related Hyper Immunoglobulin E (IgE) Syndrome (HIES), as it is characterized by elevated IgE levels, eosinophilia, and recurrent infections. The majority of HIES is caused by either autosomal dominant inheritance (AD-HIES) of mutations in the signal transducer and activator of transcriptase 3 (STAT3) gene (Grimbacher et al., 1999), or autosomal recessive inheritance (AR-HIES) primarily of mutations in the guanine-nucleotide exchange factor dedicator of cytokinesis 8 (DOCK8) gene (Su et al., 2011). However, an increased insight into the functionality of DOCK8 illuminates its impact on both the T- and B-cell compartment of the immune system, which has promoted the reclassification as a DOCK8-related combined immunodeficiency (CID). DOCK8 deficiency is a severe disorder with early onset of morbidity and high mortality rates exceeding those associated with STAT3 HIES (Aydin et al., 2015; Tsilifis et al., 2021). Since the majority of the clinical manifestations of DOCK8 deficiency pertain to the immune system, hematopoietic stem cell transplantation (HSCT) in early childhood is encouraged. However, this is challenged by the need for a HLA-matched donor and associated with adverse events such as immune rejection and graft-versus-host disease (Copelan, 2006; Aydin et al., 2019).

The ideal treatment of DOCK8 deficiency would be correcting the disease-causing mutation in the patient’s own cells, thereby restoring the DOCK8 functionality. This would circumvent the obstacle of identifying HLA compatible donors for allogeneic transplantation and eliminate the associated risks. During the past two decades, genetic therapies have shown promising results for an expanding numbers of genetic disorders (Booth et al., 2019; Porteus, 2019). Meanwhile, precise genome editing tools were developed and applied in a range of pre-clinical and even a few clinical gene therapy studies. In particular, the discovery of the CRISPR/Cas system as a highly versatile genome editing platform accelerated the development of genome editing methods (Bak et al., 2018a). This ultimately led to the 2020 Nobel Prize in Chemistry awarded to Emmanuelle Charpentier and Jennifer Doudna for ‘the development of a method for genome editing’. CRISPR/Cas offers unprecedented simplicity in facilitating genome editing, and has proven highly precise and efficient (Jensen et al., 2019; Porteus, 2019). Since the first injections of CRISPR gene edited cells into patients in 2016 (Cyranoski, 2016), there have been published reports on only a few clinical trials. These trials have marked important milestones by providing evidence on safety, whereas recent clinical trials on sickle cell disease, β-thalassemia, and transthyretin amyloidosis have been the first to demonstrate therapeutic and potentially curative potential (Frangoul et al., 2021; Gillmore et al., 2021).

In preclinical studies, CRISPR/Cas gene editing has shown tremendous potential in a wide range of diseases, but has so far not been applied to DOCK8. Here, we will elaborate on why gene editing is within the realm of possibility for treating DOCK8 deficiency. First, we briefly present our current understanding of the genetic, molecular, and cellular mechanisms involved in DOCK8 deficiency. Second, we portray the common disease manifestations and discuss current diagnostic and treatment approaches. Third, after describing recent advancements in the field of genome editing and discussing advantages and disadvantages of the different precise gene editing platforms, we define suitable CRISPR/Cas strategies for treating, which may constitute a definitive cure for DOCK8 deficiency. Finally, we give a concise summary of hurdles and challenges for using gene editing in the clinical setting.

The large DOCK8 gene is located on the short arm of chromosome 9, includes 48 exons, spans over 250 kilobases, and encodes a protein of approximately 190 kDa. Bi-allelic loss-of-function mutations in the DOCK8 gene is associated with DOCK8 deficiency (Zhang et al., 2010; Database resources of the, 2018). DOCK8 deficiency is estimated to affect less than one person per million, but the exact prevalence is unknown (Biggs et al., 2017). The disease was not recognized until 2009, and only about 200 cases have been described world-wide so far, which have been identified predominantly in populations with consanguineous marriage (Zhang et al., 2009; Biggs et al., 2017).

To get a collected overview on the different patient mutations, we performed a comprehensive data collection of 60 disease-causing DOCK8 variants described in the literature and registered in the ClinVar database. These variants are represented in Figure 1 and listed in Supplementary Table S1. Even though no specific mutation hotspot regions were identified, the majority of disease-causing mutations in DOCK8 were deletions which cover 61.5% of the variants and range from a few base pairs to deletions spanning several hundred base pairs. The high propensity for deletions has been hypothesized to be partly caused by the occurrence of repetitive genomic sequences leading to abnormal recombinations in this region (Engelhardt et al., 2009). The pathogenic variants in DOCK8 are predominantly loss of function, thus abolishing the expression of DOCK8, but occurrences of DOCK8 duplication has been shown to associate with neurodevelopmental conditions (Jing et al., 2014).

As with a few other primary immunodeficiencies, there have been reported cases of somatic reversion leading to partial re-expression of DOCK8 protein in some cell linages. These occurrences of “natural gene-therapy” may reflect the location of DOCK8 within a recombination hotspot, promoting either a somatic repair of a point-mutation, recombination-mediated gene conversion, or recombination-mediated intragenic single crossover (Pillay et al., 2021). Jing et al. observed some clinical improvement in seventeen patients with somatic reversion, with significant improvement in overall survival and age-stratified morbidity. However, these improvements were insufficient for disease elimination presumed to be due to inadequate DOCK8 re-establishment, particularly within the T cells (Pillay et al., 2021). In contrast, Pillay et al. identified three patients with biallelic compound heterozygous DOCK8 germline variants, who displayed significant DOCK8 expression in their lymphocyte subsets ranging from 10% of all B cells to 75% of all CD8⁺ T cells, while myeloid cells did not express DOCK8. DNA sequencing analyses revealed that one pathogenic allele had been genetically repaired, which was hypothesized to have occurred in either a single common lymphoid progenitor cell or a single hematopoietic stem cell. In all three patients, the somatic reversion improved survival, differentiation, and function of lymphocytes and provided great clinical improvement to the patients (Namekata et al., 2014). Such single progenitor/stem cell reversions signify that only modest DOCK8 correction frequencies by gene therapy in autologous hematopoietic stem cells or lymphoid progenitor cells could provide significant clinical benefit to the patients.

Until recently, the molecular mechanism of DOCK8 and its influence in cell homeostasis was unknown and unexplored. However, recent discoveries have shed light on these, and we present here these recent discoveries with a focus on the immunoregulatory influence of DOCK8.

The initial diagnosis of DOCK8 deficiency is based on the clinical characteristics in combination with the laboratory immunological findings, with final verification through genetic analysis. DOCK8 deficiency was described clinically for the first time in 2009 and is characterized by early-onset and severe morbidity. Cohort studies have reported around 50% probability to survive beyond 20 years of age with a mean age at death of 9–12 years (Aydin et al., 2015; Zhang et al., 2014). The disease primarily presents with atopic disease, upper and/or lower respiratory infection, frequent viral cutaneous infections, and malignancy (Figure 3) (Zhang et al., 2009; Zhang et al., 2014; Aydin et al., 2015; Haskologlu et al., 2020). Mortality occurs primarily due to infectious agents particularly affecting the skin and respiratory tracts, followed by malignancy, and less commonly CNS vasculitis (Aydin et al., 2015). Almost obligatory findings in these patients are eczema and markedly elevated IgE levels, and there is a high frequency of atopic diseases like food allergies and asthma (Chu et al., 2012; Broides et al., 2017).

Flow cytometric analysis is used to analyse intracellular expression levels of DOCK8, but it is also used to identify B cell maturation arrest and altered frequencies of CD4⁺ and CD8⁺ T cells (Caracciolo et al., 2014). The circulating peripheral blood of DOCK8-deficient patients is characterised by a decreased CD4⁺ T cell count and a shift in the CD8⁺ T cell compartment towards a more exhausted phenotypic subset (Janssen et al., 2020). The B cell compartment displays an increase in naive B cells and a reduction in memory B cells (Tang et al., 2019). However, differential counts of lymphoid cells can show a significant heterogeneity in abnormal findings, thus warranting additional clinical parameters for diagnosis (Alsum et al., 2013).

DOCK8-deficiency leads to a predisposition of cancer, particularly subtypes of haematological or epithelial origin which are often virally-induced either by Epstein Barr Virus (EBV)-driven leiomyosarcomas and lymphomas, and/or Human Papilloma Virus (HPV)-associated squamous cell carcinomas (Papan et al., 2014; Zhang et al., 2014; Aydin et al., 2015; Haskologlu et al., 2020) In the aforementioned cohort studies, 8–17% of patients had developed malignancies during the follow-up period, which included cases of lymphoma (Burkitt and non-Hodgkin lymphoma), squamous cell carcinoma, and sarcoma (Zhang et al., 2014; Aydin et al., 2015).

To summarize, physicians are encouraged to be vigilant about the clinical manifestations of DOCK8-related primary immunodeficiency. It is mainly characterized as HIES and enhances the susceptibility of recurrent viral and bacterial infection, atopic disease, and higher probability of malignancy.

Present management of DOCK8 deficiency includes frequent screening for disease progression and treatment of complications through administration of immunoprophylaxis, antiviral and antibacterial treatments prior to a definitive cure through HSCT, if a compatible donor can be identified.

The evidence supporting allogeneic HSCT for treatment of DOCK8 has been described through multiple reports (Chu et al., 2012; Aydin et al., 2019). HSCT is performed after an initial myeloablative or reduced intensity conditioning regimen consisting primarily of chemotherapy and occasionally with additional radiotherapy (Aydin et al., 2019). The purpose of conditioning is to induce adequate immunosuppression and ablation of the recipient’s hematopoietic stem cells. Administration of interferon alpha has shown efficacious as a rescue therapy for viral infections such as Herpex Simplex virus (HSV) and Human Papilloma virus (HPV) (Al-Zahrani et al., 2014; Gernez et al., 2018). In addition, patients awaiting stem cell transplantation benefit from immunoglobulin replacement therapy (IVIG) and prophylactic treatment (Bazinet and Popradi, 2019). Antibodies in DOCK8 patients have been shown to display reduced avidity, which is why IVIG is recommended despite normo- or hyperphysiological antibody levels (Janssen et al., 2014). Furthermore, prophylactic treatment is given post-transplantation to prevent infection and non-infectious complications in the period until immune reconstitution (Devetten and Armitage, 2007).

The detection and aggressive treatment of infectious disease is paramount to avert fatal progression leading to death. However, a large retrospective report consisting of 136 patients with DOCK8 immunodeficiency accentuates the severe disease progression. Unfortunately, even with early intervention with aggressive therapies or prophylaxis such as anti-bacterial, fungal, viral, immunomodulatory, and immunoglobulin replacement treatment, 63% of the patients succumb by their fourth decade of life (Aydin et al., 2015). Therefore, early allogeneic HSCT is clearly indicated, which is also the sole possibility for a curative treatment. Advancements in HSCT have vastly increased the post-HSCT survival for DOCK8 deficiency patients. Hence, patients undergoing HSCT between 1995 and 2010 had a 2-years overall survival of 57 versus 92% for patients transplanted between 2011 and 2015 (Aydin et al., 2019). However, HSCT still entails several risks of severe adverse events, particularly from haploidentical relatives or unrelated donors which pose risks of graft-versus host disease and graft failure (Fox et al., 2018). Reports show that among DOCK8 deficient patients undergoing HSCT, 33% develop acute graft versus host disease (Aydin et al., 2019). Furthermore, HSCT is not always available due to disease progression because of late or misguided diagnosis, and the lack of HLA-compatible donors (Broder et al., 2017; Slatter and Gennery, 2018; Gavrilova, 2019).

Finally, HSCT is associated with high expenses and its use is gradually increasing thus indicating the need for novel therapies that reduce the overall medical cost associated with HSCT and its side effects (Morgan et al., 2017; Mayerhoff et al., 2019). Autologous HSCT with genetically modified stem cells may constitute a promising therapy with fewer adverse outcomes and higher availability to patients with DOCK8 deficiency (Kim et al., 1996; Talib and Shepard, 2020).

Our ability to precisely rewrite and manipulate the instructions encoded in the genome has greatly expanded over the last few decades. By using programmable nucleases, such as Zinc Finger Nucleases (ZFNs) (Miller et al., 2007; Szczepek et al., 2007; Christian et al., 2010), TALE nucleases (TALENs) (Miller et al., 2011; Mussolino et al., 2011; Jinek et al., 2012) and RNA-guided Cas nucleases (Cong et al., 2013; Tebas et al., 2014), researchers can direct the creation of double-strand breaks (DSBs) to specific sites in the DNA and hereby exploit the cellular DNA repair machinery to introduce desired genetic modifications. Induced DSBs are repaired through either Non-Homologous End-joining (NHEJ), an error-prone repair mechanism that induces insertion or deletions (INDELs) at the DNA breakpoint, or Homology-Directed Repair (HDR), a precise repair pathway that uses homologous repair templates to copy from during repair of the DSB, allowing the inclusion of foreign DNA sequences into a specific locus (Figure 4A).

Using programmable nucleases, gene disruption or remodeling of regulatory sequences can be easily achieved by NHEJ. These are strategies that have already been used in clinical trials with promising results (Bushman, 2007; Xu et al., 2019; Stadtmauer et al., 2020; Frangoul et al., 2021; Gillmore et al., 2021). However, for most autosomal recessive PIDs, the addition, replacement, or correction of the affected gene is required thereby necessitating the use of the HDR pathway. This can be achieved by direct correction of the genetic mutation or through the integration of the complete or partial open reading frame cDNA sequence either directly after the endogenous promoter or into a safe harbor site in the genome with a heterologous promoter.

The ability of hematopoietic stem cells (HSCs) to perpetually self-renew and differentiate into all hematopoietic lineages makes them an ideal therapeutic target for gene therapy for treating blood and immune system diseases, including PIDs. Several gene therapy clinical trials have been performed in HSCs since the first one in 1990, and they have mainly been performed using retroviral vectors that integrate the transgene into the chromosomes of HSCs in a semi-random fashion (Booth et al., 2016). Despite clinical trials for several PIDs showing curative potential of these gene therapies, there are also multiple reports of patients developing leukemia due to insertional mutagenesis caused by the retroviral vector (Dever et al., 2016) These events were caused by vector integration close to cancer-related genes such as LMO2 and transactivation of these genes by the strong viral promoter/enhancer elements present in the retroviral vectors. Although lentiviral vectors with self-inactivating mechanisms have proven a safer alternative, they have not entirely eliminated the risks associated with random vector integration, thereby rationalizing the pursuit for safer alternatives such as precise genome editing. Undoubtedly, the advent of the CRISPR/Cas gene editing system supports the onward march towards precision gene therapy.

Initially the CRISPR/Cas system was comprised of the Cas9 endonuclease and two small RNAs: CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA). While crRNA confers specificity to a complementary region in the genome and thereby serves to guide Cas9 to its target, the tracrRNA acts as a Cas9 binding handle to enable the formation of a ribonucleoprotein (RNP) complex. In seminal work from the 2020 Nobel prize winners Jennifer Doudna and Emmanuelle Charpentier they merged the crRNA and tracrRNA into a single guide molecule (sgRNA) thereby reducing the system from three to two components (Cong et al., 2013).

The therapeutic potential of targeted gene editing in long-term repopulating HSCs (LT-HSCs) was first demonstrated in a preclinical study for X-linked Severe Combined Immunodeficiency (SCID-X1) using ZFNs delivered by mRNA electroporation and repair template delivery by an integration-defective lentiviral vector (IDLVs). In this study, Genovese et al. achieved targeted integration of a partial IL2RG cDNA comprising a super-exon of exons 5–8 of IL2RG into exon 5 of the endogenous IL2RG gene. Thus, transcription occurs from the endogenous IL2RG promoter and exon 4 splices with the newly inserted super-exon to generate a correct and full-lenght IL2RG reading frame, thereby providing a platform with the potential to correct all SCID-X1 IL2RG mutations downstream of exon 4. Long-term engraftment of the targeted HSCs in transplanted NSG mice was confirmed and they were also able to correct the defective IL2RG gene in HSCs from a patient with SCID-X1. The CRISPR/Cas system was similarly applied in HSCs for the first time to correct the mutation in the β-globin gene responsible for Sickle Cell Disease (SCD) (Lattanzi et al., 2021). Here, precise correction of the disease-causing mutation was performed with similar evidence of long-term engraftment in mice and reconstitution of functional β-globin (Mohrin et al., 2010). However, both studies also revealed what is now considered the largest challenge for applying precise HDR-mediated gene editing in HSCs, which is the low efficiencies of HDR-mediated editing in the long-term (LT)-HSC compartment compared to the progenitor cell population of the total CD34⁺ cells. This is mainly believed to be caused by HDR only being active in the late S and G2 phases of the cell cycle whereas NHEJ is the prevailing repair mechanism in quiescent cells (Wilson et al., 2008). This poses a paradoxical challenge in HSC-based gene editing since otherwise quiescent HSCs must be forced into cycling, but cycling is known to be associated with loss of stem cell properties (Song et al., 2016). Hence, NHEJ-focused HSC therapies have shown higher efficiencies in HSCs, confirmed in a recent clinical trial (Frangoul et al., 2021), but is generally more difficult to apply for recessive disorders where expression of a functional gene must be restored.

To enhance HDR frequencies, researchers have been working on different strategies that include the use of repair pathway-modulating small molecules that promote HDR or inhibit NHEJ (Lin et al., 2014; Chu et al., 2015; Robert et al., 2015; Nambiar et al., 2019; De Ravin et al., 2021; Fu et al., 2021), cell cycle synchronization to ensure S/G2 status upon editing (Charpentier et al., 2018; Shin et al., 2020), and the development of novel engineered Cas9 variants for example fusing HDR-promoting or NHEJ-inhibiting proteins to Cas9 (Jayavaradhan et al., 2019; Ferrari et al., 2020). New protocols also transiently inhibit the p53 pathway to achieve high percentages of HDR editing in LT-HSCs (Vavassori et al., 2021). The most advanced example of this is a recent study correcting the CD40 ligand gene (CD40LG), which has deactivating mutations in X‐linked hyper‐IgM syndrome type I (HIGM1) (Schiroli et al., 2019). Here, the authors introduce mRNA encoding a dominant negative p53 variant (GSE53) along the CRISPR/Cas9 gene editing components. Prior studies have shown that during gene editing in HSCs, DSBs and the presence of adeno-associated virus (AAV) vector genomes, used to deliver the HDR repair template, activate p53, which constrains HSC yield, proliferation, and engraftment of gene-edited HSCs (Cromer et al., 2021). With the addition of GSE53, the authors showed up to 30% CD40LG correction frequencies (cDNA integration) in LT-HSCs.

The HDR pathway also provides the possibility of replacing entire gene sequences or large genomic regions. This was recently showcased in LT-HSCs where a DNA repair template was designed in a way that the copy-paste mechanism replaced the one pf the α-globin genes (HBA1) with that of β-globin. This approach could prove therapeutic in patients with β-thalassemia to normalize the balance between α and β chains, thus restoring adult hemoglobin functionality (Maresca et al., 2013). Moreover, this study showed that whole gene replacement is possible in LT-HSCs, thereby providing an additional genome editing strategy for genetic diseases.

As alternatives to HDR, novel gene correction approaches based on HDR-independent targeted gene integration (Sakuma et al., 2016; Yao et al., 2017; Porto et al., 2020), base editing (Anzalone et al., 2019), and more recently prime editing (Newby et al., 2021) try to overcome the inherent limitations of HDR-mediated genome editing. In base and prime editing, the functional properties of Cas9 can be extended by fusing new effector domains to catalytically inactive Cas9 protein or Cas9 nickase. In this way, without requiring DSBs or donor DNA templates, Base Editors (BEs) mediate single-nucleotide conversions in a targeted manner, while Prime Editors (PEs) write new genetic information into a specific nicked locus directed by a small template present on the sgRNA (Figures 4B,C) (Newby et al., 2021). These are promising alternatives for genome editing, and BEs have already shown as high as 68% base editing of the β-globin gene in human LT-HSCs evaluated 16 weeks after transplantation into mice (Vaidyanathan et al., 2018) Similar evidence for PEs must be provided to reinforce their applicability in HSCs.

Retroviral gene therapy in autologous HSCs has provided clinical benefit in several PIDs including SCID-X1 (Aiuti et al., 2002; Gaspar et al., 2004; Hacein-Bey-Abina et al., 2010; Gaspar et al., 2011), Adenosine deaminase deficiency (ADA-SCID) (Aiuti et al., 2009; Boztug et al., 2010; Cicalese et al., 2016; Scott et al., 2017), Wiskott Aldrich Syndrome (WAS) (Ott et al., 2006; Aiuti et al., 2013; Hacein-Bey Abina et al., 2015), and X-linked chronic granulomatous disease (X-CGD) (Cavazzana-Calvo et al., 2010; Kang et al., 2010). However, integration of the transgene in these approaches occurs semi-randomly into the patient’s genome, and as mentioned earlier this can lead to oncogenic transformation due to insertional mutagenesis. Even vectors with lower genotoxic potential, such as lentiviral vectors (LVs), can give rise to insertional mutagenesis, which is a risk that needs to be considered in clinical applications (Brown et al., 1998; Modlich et al., 2009). Furthermore, transgene expression levels often differ from the physiological levels of the affected gene due to the use of a constitutive promoter that does not allow tissue-specific or temporal regulation of expression. Unregulated gene expression can for some diseases be detrimental exemplified by the CD40LG gene, which must be expressed at controlled levels as evidenced by preclinical mouse studies where ex vivo gene therapy in a mouse model of X-linked hyper-IgM syndrome with CD40LG-encoding murine gamma-retroviral vectors in HSCs led to lymphoproliferative disorder assumingly as a consequence of unregulated expression of the CD40L transgene (Glessner et al., 2017). DOCK8 deficiency has not been approached with retro- or lentiviral gene delivery, but copy number variation analyses have identified DOCK8 duplications to be significantly associated with a spectrum of neuropsychiatric disorders (Jing et al., 2014). Even though a direct link from DOCK8 CNV to immunological defects has not been established, this might suggest that elevated levels of DOCK8 gene expression can impede normal cellular function. Tight DOCK8 expression in different mature immune cell subsets and regulated DOCK8 expression during hematopoiesis would be impossible to establish with LVs carrying constitutive heterologous promoters and would require full delineation of the regulatory mechanisms that govern DOCK8 expression and reconstruction of a DOCK8 promoter suitable for LV use. Hence, LV-mediated gene delivery may not be a therapeutic option in DOCK8 deficiency, whereas precise gene editing approaches may be optimally suited for such diseases. In the following section, we summarize essential considerations for gene editing and describe different gene editing strategies and their potential use for correcting DOCK8-mediated immunodeficiency.

In the near future, we expect several genetic diseases such as DOCK8 deficiency, which lack efficient and safe treatment regimens, to be treatable with appropriate gene therapy strategies. The great advance compared to conventional pharmaceutical approaches is that gene therapy directly corrects the underlying genomic abnormality of the disease. This provides the possibility to cure these diseases rather than treat symptoms. Given the vast diversity in gene-editing tools, we emphasize the requirement of selecting proper therapeutic strategies that match the underlying genetics of the disease. These strategies have distinct advantages, limitations, and potential adverse effects and for many indications the choices might not be straight-forward. Therefore, when pursuing a curative treatment for DOCK8 deficiency it is imperative that several modalities are explored at the developmental stage to maximize the therapeutic effects while considering disease mechanism, mutation locations, and strategies for delivery and correction. We suggest that efficient and patient-universal correction may be achieved by exploiting the HDR pathway. Additionally, ex vivo introduction of repair templates using AAV donor templates currently seems to be the most proper delivery mechanism and may be designed to replace single or multiple exons with hot-spot mutations or perhaps even include a full cDNA through a two-step HDR strategy. However, further experimental work is warranted to evaluate the efficiency of the different strategies in pursuit of a definitive cure for DOCK8 deficiency. Meanwhile, intensive research in genome editing leads to a continuous emergence of technologies that are bound to enable future breakthroughs in clinical gene therapy. Already, CRISPR/Cas9-based clinical trials have proven successful for sickle cell disease, beta-thalassemia, and transthyretin amyloidosis (Frangoul et al., 2021; Gillmore et al., 2021), and many efforts are focused on optimizing the conditioning regimen, such as the development of anti-CD117 antibodies that deplete HSCs in a targeted and safe manner (Kwon et al., 2019). With these advances and accumulation of experience from clinical trials, the future looks bright for bringing more CRISPR/Cas9-based gene therapies to patients that are safer and more efficient.