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Review Article
CRISPR/Cas9 applications in gene therapy for
primary immunodeficiency diseases
Suk See De Ravin and Julie Brault
Laboratory of Clinical Immunology and Microbiology, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, U.S.A.
Correspondence: Suk See De Ravin (sderavin@niaid.nih.gov)
Primary immunodeficiency diseases (PIDs) encompass a range of diseases due to mutations
in genes that are critical for immunity. Haploinsufficiency and gain-of-function mutations are
more complex than simple loss-of-function mutations; in addition to increased susceptibility
to infections, immune dysregulations like autoimmunity and hyperinflammation are common
presentations. Hematopoietic stem cell (HSC) gene therapy, using integrating vectors, pro-
vides potential cure of disease, but genome-wide transgene insertions and the lack of
physiological endogenous gene regulation may yet present problems, and not applicable in
PIDs where immune regulation is paramount. Targeted genome editing addresses these
concerns; we discuss some approaches of CRISPR (Clustered Regularly Interspaced Short
Palindromic Repeats)/Cas system applicable for gene therapy in PIDs. Preclinical repair of
gene mutations and insertion of complementary DNA restore endogenous gene regulation
and they have shown very promising data for clinical application. However, ongoing studies
to characterize off-target genotoxicity, careful donor designs to ensure physiological expres-
sion, and maneuvers to optimize engraftment potential are critical to ensure successful
application of this next-gen targeted HSC gene therapy.
Introduction
Primary immunodeficiency diseases (PIDs) encompass a range of disorders due to mutations in genes
that are critical for immune function and result in absent, non-functioning or abnormally active
immune cells. Over 300 genes have been associated with PIDs [1] and, with the availability of high-
throughput next-generation sequencing, the list is rapidly growing longer. Better characterization
of genetic defects provides the opportunity to design specific gene correction strategies for the treatment
of PIDs, thus paving the way to precision medicine. In addition to delineating mutation types in PIDs,
such as point mutations, chromosomal alterations (deletions, insertions, translocations) or copy number
variation, mechanistic understanding of the genetic end products is also crucial as loss-of-function
(LOF), gain-of-function (GOF), haploinsufficiency, or dominant negative phenotypes probably require
different approaches tailored to each scenario when contemplating treatment strategies [2].
PIDs most commonly present with increased susceptibility to infections and evidence of immune dys-
regulation that may present as autoimmunity or hyperinflammation.Manyrecentlydefined PIDs
include GOF mutations in genes that affect cellular signaling pathways or immune regulation. We will
discuss the versatile applications of targeted genome editing using the CRISPR (Clustered Regularly
Interspaced Short Palindromic Repeats)/Cas system to both LOF PIDs and more challenging GOF PIDs.
The clinical manifestations of the PID may influence the approaches for treatment of the disease.
Previously well-described LOF mutations, such as those leading to severe combined immunodeficiency
(SCID) due to IL2RG defects (X-linked SCID or SCID-X1) or adenosine deaminase deficiency (ADA–
SCID), or chronic granulomatous disease (CGD), present early with severe infections. In contrast, some
recently described PIDs with abnormalities in cellular immune signaling pathways may be diagnosed
later in life with significant immune dysregulation presenting as autoimmunity [3,4]. Clinical manage-
ment of PIDs with increased susceptibility to infections has included lifelong anti-microbial prophylaxis,
Version of Record published:
23 May 2019
Received: 19 March 2019
Revised: 9 May 2019
Accepted: 13 May 2019
© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology 1
Emerging Topics in Life Sciences (2019)
https://doi.org/10.1042/ETLS20180157
immunoglobulin supplementation and, generally, consideration of hematopoietic stem cell (HSC) transplant
(HSCT) from a human leukocyte antigen-matched allogeneic donor. For patients with defects in immune cell
signaling, medical intervention can be tailored to enhance regulatory pathways or to inhibit inflammatory signals
[5–8]. The design of genome-editing approaches to treat PID, therefore, requires consideration of the specific
causative genetic mutation as well as the clinical presentation and condition of the patient.
The first successful allogeneic HSCT of a SCID-X1 patient over 50 years ago demonstrated that replacement with
normal HSCs can cure this PID [9]. Although potentially curative, allogeneic HSCT still suffers from the following
problems: potential for graft versus host disease, graft rejection, toxicities related to chemotherapeutic or immunosup-
pressive agents and most critically, the availability of matched allogeneic donor grafts [10,11]. HSC gene therapy,
however, can circumvent some of these problems by correction of autologous HSCs by insertion of a therapeutic
gene into the chromosomal DNA of the HSC that engrafts and differentiates ideally with normal function. The inser-
tion of the therapeutic gene relies on integrating vectors that deliver and insert one or more copies into the HSC.
Using γ-retroviral vectors, initial HSC gene therapy trials demonstrated significant clinical benefits, especially where
the insertion conferred a survival advantage to gene-corrected cells [12–14]. Unfortunately, lymphoid and/or
myeloid insertional mutagenesis was observed in some PIDs including SCID-X1, CGD, and Wiskott–Aldrich syn-
drome (WAS) [15–17]. More recently, human lentivirus vectors have been used, with improved safety features and
an integration pattern in the chromosomal DNA that does not favor promoters and enhancers, to reduce the risk of
oncogenic activating integrations [18]. Replication-deficient lentivectors have been examined in clinical trials for the
treatment of PIDs, including SCID-X1, CGD, WAS, ADA–SCID with some positive results [19–22]. Lentivector
gene therapy products for ADA–SCID, SCID-X1, and CGD are soon likely to become approved drugs [23,24]. The
unequivocal clinical benefits following HSC gene therapy provide further incentive to design better strategies for spe-
cificgenemodification of autologous HSCs for transplant to treat PIDs. Lentivector HSC gene therapy may yet suffer
from two disadvantages (the potential long-term genotoxicity of the quasi-random genome-wide vector integrations
and the lack of physiological regulation of gene expression) that may be eliminated by specifically targeted
gene-editing techniques.
Targeted genetic modification may offer the greatest potential yet for definitively curing the widest spectrum
of PIDs. Recent advances in nucleases have made possible precise genome editing by the use of programmable
endonucleases including zinc finger nucleases, transcription activator-like effector nucleases, or megaTALs and
more recently, the CRISPR/Cas9 system [25–31]. These genome engineering systems share an ability to recog-
nize and target specific genomic DNA sequences and create a site-specific double-strand break (DSB) or a
Figure 1. Overview of the gene-editing therapy for primary PIDs.
Autologous hematopoietic stem and immune cells apheresed from PID patients processed in vitro with CRISPR/Cas9
gene-editing tools may be administered fresh or cryopreserved following validation of product for correction efficiency, safety
and hematopoietic function before infusion.
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Emerging Topics in Life Sciences (2019)
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single-strand ‘nick’in the DNA, thus stimulating the endogenous cellular repair machinery to bring about the
genomic alteration. The main repair pathways are the error-prone non-homologous end joining (NHEJ) that
results in insertions and/or deletions (indels) at the site of the break, and the homology-directed repair (HDR)
that uses the sister chromatid or an exogenous DNA donor template to repair the break [32]. In the context of
PIDs, correction of genetic defects most frequently relies on HDR, although the NHEJ repair pathway can be
harnessed to functionally inactivate a targeted locus (‘knock-out’) by introducing small indels.
We focus our discussion on the use of the CRISPR/Cas9 system as a representative of this class of
gene-editing tools and on the genome-editing strategies that can be applied to potential cures of specific PIDs.
Although gene-edited HSC may be the best definitive therapy, for some PIDs, for example in CD40L deficiency
causing Hyper IgM syndrome, correction of primary immune cells, e.g. T-cells, may be sufficient for control
of disease, either in isolation or to improve control of disease to reduce risks of definitive transplant, whether
allogeneic or autologous gene-modified HSCs (Figure 1)[33,34].
CRISPR/Cas9 tools for gene editing
In addition to its versatility, the wide availability of CRISPR/Cas9 reagents has resulted in the rapid develop-
ment of new tools, as summarized in Figure 2, and growing knowledge leading to creative solutions to solve
the range of genetic mutations in PIDs. We present here some tools available to date and illustrate their use in
specific PIDs.
The CRISPR/Cas9 system relies on the recognition of a specific genomic DNA target complementary to a short
guide RNA sequence (gRNA, generally ∼20 nucleotides), a protospacer, adjacent to an appropriate protospacer adja-
centmotif(PAM).TheformationofanRNA–DNA pairing induces a conformational change in the Cas9 nuclease
that promotes its nuclease activity and the generation of a double-strand cleavage 3 bp upstream of the PAM
sequence [35]. The first described Cas9 nuclease, a class 2 type II CRISPR/Cas system derived from Streptococcus
pyogenes (SpCas9), recognizes a 50-NGG-30PAM sequence theoretically found every 8–12 bp in the genome
[26,31,36]. Excellent works in the field have created and uncovered Cas9 variants requiring different PAM sequences
[35,37] and nucleases from alternate bacterial species [Streptococcus thermophilus (PAM = 50-NNAGAAW-30and
Figure 2. CRISPR/Cas9 system for targeted genome editing.
The steps for CRISPR/Cas9 gene-editing tool selection from an array of CRISPR/Cas9 components are summarized (a), delivery
methods (b), and approaches harnessing HDR for gene correction (c). LHA: left homology arm; RHA: right homology arm.
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50-NGGNG-30for CRISPR1 and 3, respectively, every 64 bp) or Neisseria meningitidis (PAM = 50-NNNNGATT-30
every 128 bp)] [25,38,39] have greatly extended the scope of targetable genomic sites.
The safety profile of the CRISPR/Cas system can be improved in many ways, most simply with the use of
engineered SpCas9 High Fidelity variants [40,41]. Alternatively, studies using Cas9 nickases (Cas9n; D10A or
H840A mutations) [26,42] or catalytically inactive Cas9 (fCas9) or dead Cas9 (dCas9) fused to a FokI nuclease
domain [43–45] reported improved specificity by creating a DSB between the two binding sites. Cas9n can
improve specificity by up to 1,500-fold compared with the wild-type (WT) Cas9 with fewer off-target cleavage
events [46,47]. Also reported to possess greater specificities are Cas orthologs of the class 2 type V CRISPR/Cas
system like Cpf1/Cas12a and Cas12b nucleases from other bacterial species [48–52] compared with S. pyogenes
Cas9. Ingenious base editing approaches that convert mutants to WT sequence without DNA cleavage are
thought to limit indels at target sites. Current strategy fuses Cas9 with a cytidine or adenosine deaminase to
cause an irreversible conversion of C:G to T:A or A:T to G:C, respectively, without DNA cleavage [53–56]. The
current limitations of this approach are the correction efficiency and a relatively limited scope of targetable
mutations. Furthermore, more sensitive approaches revealed substantial off-target effects, with a 20-fold higher
off-target frequency with cytosine base editors compared with adenosine base editors [57].
The next critical component of the CRISPR genome-editing system is the guide. Initially, the length of guide
RNAs was around 100 nucleotides (spacer of 20 nucleotides complementary to the target genomic sequence) and
they were generally produced by in vitro transcription. The use of single-guide RNA (sgRNA) with a shorter
spacer of 17, 18, or 19 nucleotides improves specificity but may reduce the efficiency of genome-editing rates [58],
and requires careful evaluation for each PID. Production of gRNA by in vitro transcription may be challenging for
some groups and prohibitory for large-scale clinical grade manufacturing. Synthetic sgRNAs have potential advan-
tages including chemical modifications that increase the stability of the sgRNA and genome-editing frequencies
[59,60]. Recently, many have found Cas9 nuclease complexed with sgRNA to form a ribonucleoprotein (RNP) that
supports earlier editing activity with less associated toxicity an attractive option (Figure 2a)[40,61,62].
Since the donor for the gene-editing system determines the end product, this component of the
genome-editing system requires careful attention and will be discussed next in greater detail in the context of
specific mutations and PIDs. Donors that have been used include single-stranded oligodeoxynucleotide
(ssODN) [63,64], long ssDNA and linear or circular dsDNA [65–68], or complementary DNA (cDNA) within
an engineered virus cassette such as integrase-defective lentivirus (IDLV; transient expression and weak integra-
tion capability) [69] or recombinant adeno-associated virus (rAAV), depending on the size of the donor
(Figure 2). The rAAV as a donor delivery tool has low immunogenic potential and has high infectious capabil-
ity even in non-dividing cells and low integration rates in the genome of the host cell [70–72]. They are gener-
ally capable of delivering up to 4.5–5 kb but by using two co-transduced AAV vectors, DNA cargos of up to
6.5 kb have been reported [73]. For targeting human HSCs, rAAV serotype 6 was shown to have the greatest
affinity, and a mutant AAV6 capsid provided further improvement [74–77]. In general, donors should have
homology arms of 400 bp, codon optimization of the cDNA and modification of the PAM sequence to prevent
re-cutting of the donor or gene-modified cells (Figure 2c)[75].
CRISPR/Cas9 approaches for HSC genome editing in PIDs
Two main considerations in determining a genome-editing strategy for PIDs are firstly, the mutation size
(single or few nucleotides versus large deletions) and frequency (mutation hotspots versus disease-causing
mutations distributed throughout the gene) that affect the choice of donor template, and secondly, the func-
tional effect of the mutation. While LOF mutation can be boosted by correction of 1 or 2 alleles, diseases that
result from heterozygote mutations are challenging due to the risks of unintended cuts on the normal allele
that results in disease, or the residual effects of uncorrected mutant allele. Ideally, in haploinsufficiency muta-
tions, one needs correction of the mutant allele without disrupting the other allele. However, in GOF muta-
tions, the goal is to knock-out or repair the mutant allele while avoiding unintended cuts of the other allele.
Great work in the design of broader choices for nucleases and with improved specificity may provide the neces-
sary tools necessary for correction of these more challenging mutations [37,78,79].
LOF mutations that involve a single or few nucleotides are amenable to the use of a short oligonucleotide donor
to repair the mutation resulting in a normal genetic sequence, i.e. ‘gene repair’(Figure 2c). About two-thirds of
human genetic diseases arise from single-base mutations. To correct such mutations in situ,thesimplestapproach
is to use a short donor template, such as a ssODN [80–85] although linear or plasmid dsDNA donors have also
been used [86,87]. Various studies have evaluated the effects of the length, sense, and symmetry of the donor
© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology4
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ssODN as well as chemical modifications (such as phosphorothioate linkages both 50and 30ends) to induce higher
HDR efficiency [60,81,86,88]. This strategy was employed to repair a relatively frequent mutation in the CYBB
gene that encodes for gp91
phox
, a critical catalytic subunit of the phagocyte NADPH oxidase complex, which
results in X-linked chronic granulomatous disease (X-CGD). Using a chemically modified 100 bp ssODN with
symmetrical homology arms, delivered by electroporation with Cas9 mRNA and in vitro transcribed sgRNA, in
vitro correction rates of 30–40% of HSC were achieved that, upon transplantation into NSG immunodeficient
mice, resulted in 15–20% stable corrected cells at 5 months [80]. Subsequent improvements to this system have
included the use of chemically modified synthetic guides and non-targeting donor oligos that improved in vitro
correction rates to 50–60% (unpublished). This level of correction is sufficient to reverse the disease phenotype of
X-CGD as it relates to susceptibility of infections only. In contrast with some gene therapy approaches using
exogenous promoters, the gene-edited corrected cells restored normal physiological regulation with normal
amounts of protein expression in the appropriate mature differentiated phagocytes and functionally normal
amounts of NADPH oxidase activity. Although consideration of individual mutations seems daunting at present, it
is possible that with the rapid progress in producing synthetic guides and donor oligonucleotides, an individual
mutation-specific repair system can be optimized for treatment within a few months for patients not needing emer-
gency intervention. Optimization of this approach may provide proof-of-concept that gene-repaired HSCs can
restore HSC compartment for gene therapy in other diseases, where a single LOF mutation may account for most
or all patients, such as sickle cell disease [89]orp47
phox
-deficient CGD [90]. Of note, the level of correction for
reversal of disease phenotype for different LOF mutations likely varies and is important to define in the preclinical
studies to provide the rationale for treatment. For example, diseases, such as SCID-X1 in which functional cells
have survival advantage, require lower levels of (∼10%) correction to reverse disease phenotype [91].
While short donor oligos may be ideal for small mutations, repair of large deletions or insertions is not pos-
sible with this approach. Instead, an alternative strategy to address large mutations or simply address mutations
located widely in the gene is to insert a functional cDNA into a targeted site, generally downstream of the
endogenous promoter to restore physiological regulation (Figure 2c). Targeted integration (TI) near the start
site could potentially address > 95% of the mutations. This ‘universal’or ‘one-size-fits-all’approach has been
shown to achieve excellent rates of TI of the cDNA, or portions of genes at a targeted site (a ‘knock-in’) where
the final genomic sequence is altered to provide a functional gene product under the control of the endogenous
promoter. Although the donors can be delivered many ways, including IDLV [83,92], the best outcomes are
observed following delivery by rAAV. Very encouraging preclinical data pave the way for imminent clinical
application for treatment of SCID-X1 due to IL2RG gene defects [91–93] and CD40L for XHIM [34,94]. Other
PIDs reporting encouraging results following targeted knock-ins include BTK for X-linked agammaglobuline-
mia (XLA) [95] and CYBB for X-CGD [76,96,97].
Recent advances in optimizing the CRISPR/Cas system have generally resulted in excellent TI rates over 50% in
vitro. Critical factors to be taken into consideration to ensure successful clinical outcomes for gene-edited HSC gene
therapy include an evaluation of the functional end product ( protein expressed) as well as the engraftment capabilities
following CRISPR/Cas9 gene editing. In addition to endogenous gene regulation, factors such as the Kozak consensus
sequence, intronic regulatory elements, and polyadenylation length ( poly(A)) should be taken into consideration
when designing donors (Figure 2). To illustrate, we evaluated the ‘knock-in’approach targeting the insertion of a
partial cDNA (CYBB Exon 7–13) using our previously reported guide targeting CYBB E7 c.676 with the ssODN gene
repair correction described above [98]. Expression of the donor encoding CYBB Exons 7 to 13 is regulated by the
endogenous CYBB promoter. We observed significantly reduced amounts of gp91
phox
protein per cell produced that
was independent of TI rates (>50–70%). Substituting the poly(A) with a longer poly(A) significantly improved the
amount of protein produced. Similarly, the addition of Woodchuck Hepatitis Virus Post Transcriptional Regulatory
Element (WPRE) significantly increases expression of genes delivered by viral vectors [94,99,100]. Furthermore, there
may be critical regulatory elements in introns lost with cDNAs that are devoid of all introns. This was illustrated by
Sweeney et al. who showed that TI in CYBB E1 with a CYBB E1–13 cDNA failed to restore the gp91
phox
expression,
while the expression was restored when the donor was inserted in Exon 2, suggesting the presence of critical regula-
tory elements in Intron 1 [98]. This problem may be circumvented by the retention of the critical intronic sequences
that may also allow the expression of isoforms under the physiological regulation of the promoter.
The engraftment capabilities of gene-edited HSCs need careful evaluation. We previously observed good engraft-
ment rates of 30–40% following transplants of ∼1 million CRISPR/Cas9/ssODN gene-repaired HSCs per mouse
(NOD SCIDyc-deficient strain) conditioned with 20 mg/kg busulfan. Our current experiences with AAV-mediated
gene ‘knock-in’approaches have observed lower engraftment rates in general (unpublished). Several groups have
© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology 5
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reported immune responses to viruses such as interferon-inducible antiviral factors or activation of p53-mediated
DNA damage response induced by DSBs which may alter stemness of the HSCs and impair the ability to engraft
[101–104]. Better understanding of the mechanisms involved may lead to discovery of agents that may counteract
these responses, such as Cyclosporin H [105,106], or agents that enhance HSC self-renewal such as
pyrimido-indole derivative UM171 and aryl hydrocarbon receptor antagonist StemRegenin 1 [77,107,108].
Since HDR occurs most efficiently during the S/G2 phases of the cell cycle when DNA is relaxed and more access-
ible [109], there are molecules targeting factors critical for DNA repair mechanisms that may also promote HDR.
Synchronizing Cas9 expression with activation of the HDR machinery may also enhance templated repair [110–112]
or by the use of inhibitors of key enzymes of the NHEJ pathway like SCR7, an inhibitor of the DNA ligase IV
DNA-binding domain, or an inhibitor of 53BP1 (i53) [113,114]. A 50–70% increase in gene correction rates in vitro
(both gene repair and targeted gene knock-in) was achieved with the use of the inhibitor of 53BP1 (unpublished).
Long-term safety and efficacy studies of NSG transplants for gene-modified HSCs with the aid of i53 are ongoing.
GOF PIDs include recently characterized PIDs, such as phosphoinositide-3-kinase δ-syndrome (PIK3CD), or
cytotoxic lymphocyte antigen-4 (CTLA-4) haploinsufficiency with pathological increased cellular signaling and
hyperactivation, resulting in clinical disease [115–119]. In PIDs due to GOF mutations, transplants from allo-
geneic donors have shown that strategies that result in chimeric populations of gene-mutant cells and genetic-
ally normal cells will likely not reverse patient’s clinical phenotype. Strategies to cause new mutant alleles may
result in mutant phenotype, while TI into the normal allele will not alleviate the end product due to the
mutant allele. There are challenging strategies that at least in theory may revert phenotype, such as targeted
knock-out of the mutant allele and replacing the mutant allele with a TI of normal cDNA. Single nucleotide
GOF mutations may also benefit from specific base editing that corrects the specific mutation without risks of
DSB that potentially creates new mutants.
Conclusion
In conclusion, the CRISPR/Cas9 technology has transformed biomedical research and preclinical data strongly
support the translation of specific genome editing for the treatment of a variety of primary immunodeficiencies.
There are critical ongoing research efforts to develop more sensitive techniques to detect chromosomal altera-
tions due to unintended effects of endonucleases. Thorough evaluation of engraftment capabilities of
gene-edited HSCs particularly with AAV-delivered donors and detailed analysis of functional outcomes for
each corrected gene defect is essential for effective correction of PIDs. Despite some concerns for off-target
indels, the potential for circumventing genome-wide vector integration concerns, and to restore physiological
control under endogenous promoters should greatly improve the safety of HSC gene therapy. Continued pro-
gress in gene-editing tools will hopefully provide broader range of choices for correcting currently challenging
PIDs, especially those due to GOF mutations.
Summary
•HSC gene therapy is clinically beneficial for some PIDs.
•Targeted genome editing can avoid genome-wide integration and constitutive expression of
transgene.
•CRISPR/Cas approach to repair gene mutations or insertion of cDNA at endogenous gene
locus can restore physiological regulation.
•Preclinical CRISPR/Cas9 studies for some LOF mutation PID are ready for translation to the
clinic.
•Future direction —CRISPR/Cas9 is versatile and may provide innovative tools and strategies
to address the range of genetic mutations in PIDs, such as haploinsufficiency, dominant
negative or GOF mutations.
© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology6
Emerging Topics in Life Sciences (2019)
https://doi.org/10.1042/ETLS20180157
Abbreviations
ADA, adenosine deaminase deficiency; cDNA, complementary DNA; CGD, chronic granulomatous disease;
CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; DSB, double-strand break;
GOF, gain-of-function; HDR, homology-directed repair; HSC, Hematopoietic stem cell; LOF, loss-of-function;
NHEJ, non-homologous end joining; PAM, protospacer adjacent motif; PIDs, Primary immunodeficiency
diseases; rAAV, recombinant adeno-associated virus; RNP, ribonucleoprotein; SCID, severe combined
immunodeficiency; sgRNA, single-guide RNA; ssODN, single-stranded oligodeoxynucleotide; TI, targeted
integration; WAS, Wiskott–Aldrich syndrome; WT, wild-type; X-CGD, X-linked chronic granulomatous disease.
Funding
This work is supported by the Intramural Research Program of the National Institutes of Allergy and Infectious
Diseases and the National Institutes of Health.
Acknowledgements
We owe our gratitude to our patients who have volunteered and supported us in our research, our colleagues
at the Dowling Apheresis Center, and the Center for Cell Engineering at the Clinical Center for support with
research stem cell collections. We also thank Dr Kol Zarember for the helpful comments for the review.
Competing Interests
The Authors declare that there are no competing interests associated with the manuscript.
References
1 Picard, C., Bobby Gaspar, H., Al-Herz, W., Bousfiha, A., Casanova, J.L., Chatila, T. et al. (2018) International Union of Immunological Societies: 2017
Primary Immunodeficiency Diseases Committee Report on inborn errors of immunity. J. Clin. Immunol. 38,96–128 https://doi.org/10.1007/
s10875-017-0464-9
2 Lodish, H., Berk, A., Zipursky, S.L., Matsudaira, P., Baltimore, D. and Darnell, J. (2000) Genetic Analysis in Cell Biology, 4th edn, W. H. Freeman,
New York
3 Allenspach, E. and Torgerson, T.R. (2016) Autoimmunity and primary immunodeficiency disorders. J. Clin. Immunol. 36, S57–S67
https://doi.org/10.1007/s10875-016-0294-1
4 Schmidt, R.E., Grimbacher, B. and Witte, T. (2018) Autoimmunity and primary immunodeficiency: two sides of the same coin? Nat. Rev. Rheumatol. 14,
7–18 https://doi.org/10.1038/nrrheum.2017.198
5 Lee, S., Moon, J.S., Lee, C.R., Kim, H.E., Baek, S.M., Hwang, S. et al. (2016) Abatacept alleviates severe autoimmune symptoms in a patient carrying
a de novo variant in CTLA-4. J. Allergy Clin. Immun. 137, 327–330 https://doi.org/10.1016/j.jaci.2015.08.036
6O’Shea, J.J. and Plenge, R. (2012) JAK and STAT signaling molecules in immunoregulation and immune-mediated disease. Immunity 36, 542–550
https://doi.org/10.1016/j.immuni.2012.03.014
7 Schwartz, D.M., Bonelli, M., Gadina, M. and O’Shea, J.J. (2016) Type I/II cytokines, JAKs, and new strategies for treating autoimmune diseases.
Nat. Rev. Rheumatol. 12,25–36 https://doi.org/10.1038/nrrheum.2015.167
8 Weinacht, K.G., Charbonnier, L.M., Alroqi, F., Plant, A., Qiao, Q., Wu, H. et al. (2017) Ruxolitinib reverses dysregulated T helper cell responses and
controls autoimmunity caused by a novel signal transducer and activator of transcription 1 (STAT1) gain-of-function mutation. J. Allergy Clin. Immun.
139, 1629–1640 https://doi.org/10.1016/j.jaci.2016.11.022
9 Gatti, R.A., Meuwissen, H.J., Allen, H.D., Hong, R. and Good, R.A. (1968) Immunological reconstitution of sex-linked lymphopenic immunological
deficiency. Lancet 2, 1366–1369 https://doi.org/10.1016/S0140-6736(68)92673-1
10 Shamriz, O. and Chandrakasan, S. (2019) Update on advances in hematopoietic cell transplantation for primary immunodeficiency disorders.
Immunol. Allergy Clin. North Am. 39, 113–128 https://doi.org/10.1016/j.iac.2018.08.003
11 Kang, E. and Gennery, A. (2014) Hematopoietic stem cell transplantation for primary immunodeficiencies. Hematol. Oncol. Clin. North Am. 28,
1157–1170 https://doi.org/10.1016/j.hoc.2014.08.006
12 Malech, H.L., Maples, P.B., Whiting-Theobald, N., Linton, G.F., Sekhsaria, S., Vowells, S.J. et al. (1997) Prolonged production of NADPH
oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease. Proc. Natl Acad. Sci. U.S.A. 94, 12133–12138
https://doi.org/10.1073/pnas.94.22.12133
13 Cavazzana-Calvo, M., Hacein-Bey, S., de Saint Basile, G., Gross, F., Yvon, E., Nusbaum, P. et al. (2000) Gene therapy of human severe combined
immunodeficiency (SCID)-X1 disease. Science 288, 669–672 https://doi.org/10.1126/science.288.5466.669
14 Gaspar, H.B., Parsley, K.L., Howe, S., King, D., Gilmour, K.C., Sinclair, J. et al. (2004) Gene therapy of X-linked severe combined immunodeficiency by
use of a pseudotyped gammaretroviral vector. Lancet 364, 2181–2187 https://doi.org/10.1016/S0140-6736(04)17590-9
15 Hacein-Bey-Abina, S., Von Kalle, C., Schmidt, M., McCormack, M.P., Wulffraat, N., Leboulch, P. et al. (2003) LMO2-associated clonal T cell proliferation
in two patients after gene therapy for SCID-X1. Science 302, 415–419 https://doi.org/10.1126/science.1088547
16 Stein, S., Ott, M.G., Schultze-Strasser, S., Jauch, A., Burwinkel, B., Kinner, A. et al. (2010) Genomic instability and myelodysplasia with monosomy 7
consequent to EVI1 activation after gene therapy for chronic granulomatous disease. Nat. Med. 16, 198–204 https://doi.org/10.1038/nm.2088
17 Braun, C.J., Boztug, K., Paruzynski, A., Witzel, M., Schwarzer, A., Rothe, M. et al. (2014) Gene therapy for Wiskott-Aldrich syndrome–long-term efficacy
and genotoxicity. Sci. Transl. Med. 6, 227ra33 https://doi.org/10.1126/scitranslmed.3007280
© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology 7
Emerging Topics in Life Sciences (2019)
https://doi.org/10.1042/ETLS20180157
18 Zufferey, R., Dull, T., Mandel, R.J., Bukovsky, A., Quiroz, D., Naldini, L. et al. (1998) Self-inactivating lentivirus vector for safe and efficient in vivo gene
delivery. J. Virol. 72, 9873–9880 PMID:9811723
19 Aiuti, A., Biasco, L., Scaramuzza, S., Ferrua, F., Cicalese, M.P., Baricordi, C. et al. (2013) Lentiviral hematopoietic stem cell gene therapy in patients
with Wiskott-Aldrich syndrome. Science 341, 1233151 https://doi.org/10.1126/science.1233151
20 Abina S, H.-B., Gaspar, H.B., Blondeau, J., Caccavelli, L., Charrier, S., Buckland, K. et al. (2015) Outcomes following gene therapy in patients with
severe Wiskott-Aldrich syndrome. J. Am. Med. Assoc. 313, 1550–1563 https://doi.org/10.1001/jama.2015.3253
21 De Ravin, S.S., Wu, X., Moir, S., Anaya-O’Brien, S., Kwatemaa, N., Littel, P. et al. (2016) Lentiviral hematopoietic stem cell gene therapy for X-linked
severe combined immunodeficiency. Sci. Transl. Med. 8, 335ra57 https://doi.org/10.1126/scitranslmed.aad8856
22 Carbonaro, D.A., Zhang, L., Jin, X., Montiel-Equihua, C., Geiger, S., Carmo, M. et al. (2014) Preclinical demonstration of lentiviral vector-mediated
correction of immunological and metabolic abnormalities in models of adenosine deaminase deficiency. Mol. Ther. 22, 607–622
https://doi.org/10.1038/mt.2013.265
23 Aiuti, A., Roncarolo, M.G. and Naldini, L. (2017) Gene therapy for ADA-SCID, the first marketing approval of an ex vivo gene therapy in Europe: paving
the road for the next generation of advanced therapy medicinal products. EMBO Mol. Med. 9, 737–740 https://doi.org/10.15252/emmm.201707573
24 Ferrua, F. and Aiuti, A. (2017) Twenty-five years of gene therapy for ADA-SCID: from bubble babies to an approved drug. Hum. Gene Ther. 28,
972–981 https://doi.org/10.1089/hum.2017.175
25 Gasiunas, G., Barrangou, R., Horvath, P. and Siksnys, V. (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive
immunity in bacteria. Proc. Natl Acad. Sci. U.S.A. 109, E2579–E2586 https://doi.org/10.1073/pnas.1208507109
26 Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A. and Charpentier, E. (2012) A programmable dual-RNA-guided DNA endonuclease in
adaptive bacterial immunity. Science 337, 816–821 https://doi.org/10.1126/science.1225829
27 Sander, J.D. and Joung, J.K. (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355
https://doi.org/10.1038/nbt.2842
28 Jinek, M., East, A., Cheng, A., Lin, S., Ma, E. and Doudna, J. (2013) RNA-programmed genome editing in human cells. eLife 2, e00471
https://doi.org/10.7554/eLife.00471
29 Hsu, P.D., Lander, E.S. and Zhang, F. (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278
https://doi.org/10.1016/j.cell.2014.05.010
30 Doudna, J.A. and Charpentier, E. (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096
https://doi.org/10.1126/science.1258096
31 Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N. et al. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339,
819–823 https://doi.org/10.1126/science.1231143
32 Chapman, J.R., Taylor, M.R.G. and Boulton, S.J. (2012) Playing the end game: DNA double-strand break repair pathway choice. Mol. Cell 47, 497–510
https://doi.org/10.1016/j.molcel.2012.07.029
33 Hubbard, N., Hagin, D., Sommer, K., Song, Y., Khan, I., Clough, C. et al. (2016) Targeted gene editing restores regulated CD40L function in X-linked
hyper-IgM syndrome. Blood 127, 2513–2522 https://doi.org/10.1182/blood-2015-11-683235
34 Kuo, C.Y., Long, J.D., Campo-Fernandez, B., de Oliveira, S., Cooper, A.R., Romero, Z. et al. (2018) Site-specific gene editing of human hematopoietic
stem cells for X-linked hyper-IgM syndrome. Cell Rep. 23, 2606–2616 https://doi.org/10.1016/j.celrep.2018.04.103
35 Wilkinson, R.A., Martin, C., Nemudryi, A.A. and Wiedenheft, B. (2019) CRISPR RNA-guided autonomous delivery of Cas9. Nat. Struct. Mol. Biol. 26,
14–24 https://doi.org/10.1038/s41594-018-0173-y
36 Hsu, P.D., Scott, D.A., Weinstein, J.A., Ran, F.A., Konermann, S., Agarwala, V. et al. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases.
Nat. Biotechnol. 31, 827–832 https://doi.org/10.1038/nbt.2647
37 Kleinstiver, B.P., Prew, M.S., Tsai, S.Q., Topkar, V.V., Nguyen, N.T., Zheng, Z. et al. (2015) Engineered CRISPR-Cas9 nucleases with altered PAM
specificities. Nature 523, 481–485 https://doi.org/10.1038/nature14592
38 Zhang, Y., Heidrich, N., Ampattu, B.J., Gunderson, C.W., Seifert, H.S., Schoen, C. et al. (2013) Processing-independent CRISPR RNAs limit natural
transformation in Neisseria meningitidis.Mol Cell. 50, 488–503 https://doi.org/10.1016/j.molcel.2013.05.001
39 Esvelt, K.M., Mali, P., Braff, J.L., Moosburner, M., Yaung, S.J. and Church, G.M. (2013) Orthogonal Cas9 proteins for RNA-guided gene regulation and
editing. Nat. Methods 10, 1116–1121 https://doi.org/10.1038/nmeth.2681
40 Vakulskas, C.A., Dever, D.P., Rettig, G.R., Turk, R., Jacobi, A.M., Collingwood, M.A. et al. (2018) A high-fidelity Cas9 mutant delivered as a
ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat. Med. 24, 1216–1224
https://doi.org/10.1038/s41591-018-0137-0
41 Kleinstiver, B.P., Pattanayak, V., Prew, M.S., Tsai, S.Q., Nguyen, N.T. and Joung, J.K. (2016) High-fidelity CRISPR-Cas9 nucleases with no detectable
genome-wide off-target effects. Nature 529, 490–495 https://doi.org/10.1038/nature16526
42 Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E. et al. (2013) RNA-guided human genome engineering via Cas9. Science 339,
823–826 https://doi.org/10.1126/science.1232033
43 Tsai, S.Q., Wyvekens, N., Khayter, C., Foden, J.A., Thapar, V., Reyon, D. et al. (2014) Dimeric CRISPR RNA-guided FokI nucleases for highly specific
genome editing. Nat. Biotechnol. 32, 569–576 https://doi.org/10.1038/nbt.2908
44 Guilinger, J.P., Thompson, D.B. and Liu, D.R. (2014) Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome
modification. Nat. Biotechnol. 32, 577–582 https://doi.org/10.1038/nbt.2909
45 Wyvekens, N., Topkar, V.V., Khayter, C., Joung, J.K. and Tsai, S.Q. (2015) Dimeric CRISPR RNA-guided FokI-dCas9 nucleases directed by truncated
gRNAs for highly specific genome editing. Hum. Gene Ther. 26, 425–431 https://doi.org/10.1089/hum.2015.084
46 Ran, F.A., Hsu, P.D., Lin, C.Y., Gootenberg, J.S., Konermann, S., Trevino, A.E. et al. (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced
genome editing specificity. Cell 154, 1380–1389 https://doi.org/10.1016/j.cell.2013.08.021
47 Cho, S.W., Kim, S., Kim, Y., Kweon, J., Kim, H.S., Bae, S. et al. (2014) Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases
and nickases. Genome Res. 24, 132–141 https://doi.org/10.1101/gr.162339.113
48 Zetsche, B., Gootenberg, J.S., Abudayyeh, O.O., Slaymaker, I.M., Makarova, K.S., Essletzbichler, P. et al. (2015) Cpf1 is a single-RNA-guided
endonuclease of a Class 2 CRISPR-Cas system. Cell 163, 759–771 https://doi.org/10.1016/j.cell.2015.09.038
© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology8
Emerging Topics in Life Sciences (2019)
https://doi.org/10.1042/ETLS20180157
49 Kleinstiver, B.P., Tsai, S.Q., Prew, M.S., Nguyen, N.T., Welch, M.M., Lopez, J.M. et al. (2016) Genome-wide specificities of CRISPR-Cas Cpf1 nucleases
in human cells. Nat. Biotechnol. 34, 869–874 https://doi.org/10.1038/nbt.3620
50 Fagerlund, R.D., Staals, R.H.J. and Fineran, P.C. (2015) The Cpf1 CRISPR-Cas protein expands genome-editing tools. Genome Biol. 16, 251
https://doi.org/10.1186/s13059-015-0824-9
51 Teng, F., Cui, T.T., Feng, G.H., Guo, L., Xu, K., Gao, Q.Q. et al. (2018) Repurposing CRISPR-Cas12b for mammalian genome engineering. Cell Discov.
4,63https://doi.org/10.1038/s41421-018-0069-3
52 Strecker, J., Jones, S., Koopal, B., Schmid-Burgk, J., Zetsche, B., Gao, L.Y. et al. (2019) Engineering of CRISPR-Cas12b for human genome editing.
Nat. Commun. 10, 212 https://doi.org/10.1038/s41467-018-08224-4
53 Nishida, K., Arazoe, T., Yachie, N., Banno, S., Kakimoto, M., Tabata, M. et al. (2016) Targeted nucleotide editing using hybrid prokaryotic and vertebrate
adaptive immune systems. Science 353, 919–921 https://doi.org/10.1126/science.aaf7573
54 Komor, A.C., Kim, Y.B., Packer, M.S., Zuris, J.A. and Liu, D.R. (2016) Programmable editing of a target base in genomic DNA without double-stranded
DNA cleavage. Nature 533, 420–424 https://doi.org/10.1038/nature17946
55 Gaudelli, N.M., Komor, A.C., Rees, H.A., Packer, M.S., Badran, A.H., Bryson, D.I. et al. (2017) Programmable base editing of A•TtoG•C in genomic
DNA without DNA cleavage. Nature 551, 464–471 https://doi.org/10.1038/nature24644
56 Eid, A., Alshareef, S. and Mahfouz, M.M. (2018) CRISPR base editors: genome editing without double-stranded breaks. Biochem. J. 475, 1955–1964
https://doi.org/10.1042/BCJ20170793
57 Zuo, E.W., Sun, Y.D., Wei, W., Yuan, T.L., Ying, W.Q., Sun, H. et al. (2019) Cytosine base editor generates substantial off-target single-nucleotide
variants in mouse embryos. Science 364, 289–292 https://doi.org/10.1126/science.aav9973
58 Fu, Y.F., Sander, J.D., Reyon, D., Cascio, V.M. and Joung, J.K. (2014) Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat.
Biotechnol. 32, 279–284 https://doi.org/10.1038/nbt.2808
59 Moon, S.B., Kim, D.Y., Ko, J.H., Kim, J.S. and Kim, Y.S. (2019) Improving CRISPR genome editing by engineering guide RNAs. Trends Biotechnol.
https://doi.org/10.1016/j.tibtech.2019.01.009
60 Hendel, A., Bak, R.O., Clark, J.T., Kennedy, A.B., Ryan, D.E., Roy, S. et al. (2015) Chemically modified guide RNAs enhance CRISPR-Cas genome
editing in human primary cells. Nat. Biotechnol. 33, 985–989 https://doi.org/10.1038/nbt.3290
61 Dever, D.P., Bak, R.O., Reinisch, A., Camarena, J., Washington, G., Nicolas, C.E. et al. (2016) CRISPR/Cas9 β-globin gene targeting in human
haematopoietic stem cells. Nature 539, 384–389 https://doi.org/10.1038/nature20134
62 Gundry, M.C., Brunetti, L., Lin, A., Mayle, A.E., Kitano, A., Wagner, D. et al. (2016) Highly efficient genome editing of murine and human hematopoietic
progenitor cells by CRISPR/Cas9. Cell Rep. 17, 1453–1461 https://doi.org/10.1016/j.celrep.2016.09.092
63 Miura, H., Gurumurthy, C.B., Sato, T., Sato, M. and Ohtsuka, M. (2015) CRISPR/Cas9-based generation of knockdown mice by intronic insertion of
artificial microRNA using longer single-stranded DNA. Sci. Rep. 5, 12799 https://doi.org/10.1038/srep12799
64 Yoshimi, K., Kunihiro, Y., Kaneko, T., Nagahora, H., Voigt, B. and Mashimo, T. (2016) ssODN-mediated knock-in with CRISPR-Cas for large genomic
regions in zygotes. Nat. Commun. 7, 10431 https://doi.org/10.1038/ncomms10431
65 Jun, S., Lim, H., Jang, H., Lee, W., Ahn, J., Lee, J.H. et al. (2018) Straightforward delivery of linearized double-stranded DNA encoding sgRNA and
donor DNA for the generation of single nucleotide variants based on the CRISPR/Cas9 system. ACS Synth. Biol. 7, 1651–1659
https://doi.org/10.1021/acssynbio.7b00345
66 Quadros, R.M., Miura, H., Harms, D.W., Akatsuka, H., Sato, T., Aida, T. et al. (2017) Easi-CRISPR: a robust method for one-step generation of mice
carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol. 18,92
https://doi.org/10.1186/s13059-017-1220-4
67 Ma, Y.W., Zhang, X., Shen, B., Lu, Y.D., Chen, W., Ma, J. et al. (2014) Generating rats with conditional alleles using CRISPR/Cas9. Cell Res. 24,
122–125 https://doi.org/10.1038/cr.2013.157
68 Yang, H., Wang, H.Y., Shivalila, C.S., Cheng, A.W., Shi, L.Y. and Jaenisch, R. (2013) One-step generation of mice carrying reporter and conditional
alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370–1379 https://doi.org/10.1016/j.cell.2013.08.022
69 Ortinski, P.I., O’Donovan, B., Dong, X.Y. and Kantor, B. (2017) Integrase-deficient lentiviral vector as an all-in-one platform for highly efficient CRISPR/
Cas9-mediated gene editing. Mol. Ther. Methods Clin. D. 5, 153–164 https://doi.org/10.1016/j.omtm.2017.04.002
70 Miller, D.G., Petek, L.M. and Russell, D.W. (2003) Human gene targeting by adeno-associated virus vectors is enhanced by DNA double-strand breaks.
Mol. Cell. Biol. 23, 3550–3557 https://doi.org/10.1128/MCB.23.10.3550-3557.2003
71 Russell, D.W. and Hirata, R.K. (1998) Human gene targeting by viral vectors. Nat. Genet. 18, 325–330 https://doi.org/10.1038/ng0498-325
72 Khan, I.F., Hirata, R.K. and Russell, D.W. (2011) AAV-mediated gene targeting methods for human cells. Nat. Protoc. 6, 482–501
https://doi.org/10.1038/nprot.2011.301
73 Bak, R.O. and Porteus, M.H. (2017) CRISPR-mediated integration of large gene cassettes using AAV donor vectors. Cell Rep. 20, 750–756
https://doi.org/10.1016/j.celrep.2017.06.064
74 Song, L.J., Kauss, M.A., Kopin, E., Chandra, M., Ul-Hasan, T., Miller, E. et al. (2013) Optimizing the transduction efficiency of capsid-modified AAV6
serotype vectors in primary human hematopoietic stem cells in vitro and in a xenograft mouse model in vivo. Cytotherapy 15, 986–998
https://doi.org/10.1016/j.jcyt.2013.04.003
75 Bak, R.O., Dever, D.P. and Porteus, M.H. (2018) CRISPR/cas9 genome editing in human hematopoietic stem cells. Nat. Protoc. 13, 358–376
https://doi.org/10.1038/nprot.2017.143
76 De Ravin, S.S., Reik, A., Liu, P.Q., Li, L.H., Wu, X.L., Su, L. et al. (2016) Targeted gene addition in human CD34
+
hematopoietic cells for correction of
X-linked chronic granulomatous disease. Nat. Biotechnol. 34, 424–429 https://doi.org/10.1038/nbt.3513
77 Charlesworth, C.T., Camarena, J., Cromer, M.K., Vaidyanathan, S., Bak, R.O., Carte, J.M. et al. (2018) Priming human repopulating hematopoietic stem
and progenitor cells for Cas9/sgRNA gene targeting. Mol. Ther. Nucl. Acids 12,89–104 https://doi.org/10.1016/j.omtn.2018.04.017
78 Gao, L.Y., Cox, D.B.T., Yan, W.X., Manteiga, J.C., Schneider, M.W., Yamano, T. et al. (2017) Engineered Cpf1 variants with altered PAM specificities.
Nat. Biotechnol. 35, 789–792 https://doi.org/10.1038/nbt.3900
79 Slaymaker, I.M., Gao, L.Y., Zetsche, B., Scott, D.A., Yan, W.X. and Zhang, F. (2016) Rationally engineered Cas9 nucleases with improved specificity.
Science 351,84–88 https://doi.org/10.1126/science.aad5227
© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology 9
Emerging Topics in Life Sciences (2019)
https://doi.org/10.1042/ETLS20180157
80 De Ravin, S.S., Li, L.H., Wu, X.L., Choi, U., Allen, C., Koontz, S. et al. (2017) CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with
X-linked chronic granulomatous disease. Sci. Transl. Med. 9, eaah3480 https://doi.org/10.1126/scitranslmed.aah3480
81 Richardson, C.D., Ray, G.J., DeWitt, M.A., Curie, G.L. and Corn, J.E. (2016) Enhancing homology-directed genome editing by catalytically active and
inactive CRISPR-Cas9 using asymmetric donor DNA. Nat. Biotechnol. 34, 339–344 https://doi.org/10.1038/nbt.3481
82 Hoban, M.D., Lumaquin, D., Kuo, C.Y., Romero, Z., Long, J., Ho, M. et al. (2016) CRISPR/Cas9-mediated correction of the sickle mutation in human
CD34
+
cells. Mol. Ther. 24, 1561–1569 https://doi.org/10.1038/mt.2016.148
83 Hoban, M.D., Cost, G.J., Mendel, M.C., Romero, Z., Kaufman, M.L., Joglekar, A.V. et al. (2015) Correction of the sickle cell disease mutation in human
hematopoietic stem/progenitor cells. Blood 125, 2597–2604 https://doi.org/10.1182/blood-2014-12-615948
84 DeWitt, M.A., Magis, W., Bray, N.L., Wang, T., Berman, J.R., Urbinati, F. et al. (2016) Selection-free genome editing of the sickle mutation in human
adult hematopoietic stem/progenitor cells. Sci. Transl. Med. 8, 360ra134 https://doi.org/10.1126/scitranslmed.aaf9336
85 Agudelo, D., Duringer, A., Bozoyan, L., Huard, C.C., Carter, S., Loehr, J. et al. (2017) Marker-free coselection for CRISPR-driven genome editing in
human cells. Nat. Methods 14, 615–620 https://doi.org/10.1038/nmeth.4265
86 Renaud, J.B., Boix, C., Charpentier, M., De Cian, A., Cochennec, J., Duvernois-Berthet, E. et al. (2016) Improved genome editing efficiency and flexibility
using modified oligonucleotides with TALEN and CRISPR-Cas9 nucleases. Cell Rep. 14,2263–2272 https://doi.org/10.1016/j.celrep.2016.02.018
87 Song, F. and Stieger, K. (2017) Optimizing the DNA donor template for homology-directed repair of double-strand breaks. Mol. Ther. Nucl. Acids 7,
53–60 https://doi.org/10.1016/j.omtn.2017.02.006
88 Yang, L., Guell, M., Byrne, S., Yang, J.L., De Los Angeles, A., Mali, P. et al. (2013) Optimization of scarless human stem cell genome editing. Nucl.
Acids Res. 41, 9049–9061 https://doi.org/10.1093/nar/gkt555
89 Uchida, N., Li, L., Haro-Mora, J.J., Demirci, S., Nassehi, T., Gamer, J. et al. (2018) Development of a clinically applicable method for high-efficiency
gene correction of plerixafor-mobilized CD34+ cells from patients with sickle cell disease.ASH Annual Meeting, San Diego, CA
90 Merling, R.K., Kuhns, D.B., Sweeney, C.L., Wu, X., Burkett, S., Chu, J. et al. (2017) Gene-edited pseudogene resurrection corrects p47
phox
-deficient
chronic granulomatous disease. Blood Adv. 1, 270–278 https://doi.org/10.1182/bloodadvances.2016001214
91 Schiroli, G., Ferrari, S., Conway, A., Jacob, A., Capo, V., Albano, L. et al. (2017) Preclinical modeling highlights the therapeutic potential of
hematopoietic stem cell gene editing for correction of SCID-X1. Sci. Transl. Med. 9, eaan0820 https://doi.org/10.1126/scitranslmed.aan0820
92 Genovese, P., Schiroli, G., Escobar, G., Di Tomaso, T., Firrito, C., Calabria, A. et al. (2014) Targeted genome editing in human repopulating
haematopoietic stem cells. Nature 510, 235–240 https://doi.org/10.1038/nature13420
93 Pavel-Dinu, M., Wiebking, V., Dejene, B.T, Srifa, W., Mantri, S., Nicolas, C.E et al. (2019) A safe and efficient universal genome targeting therapeutic
approach for SCID-X1 in human long-term hematopoietic stem cells. Nat. Commun. 10, 2021 https://doi.org/10.1038/s41467-019-09614-y
94 Hubbard, N.W., Hagin, D., Sommer, K., Song, Y.M., Clough, C., Khan, I.F. et al. (2016) Targeted gene editing restores regulated CD40L expression and
function in X-Higm T cells. Blood 127, 2513–2522 https://doi.org/10.1182/blood-2015-11-683235
95 Maerken, M., MacEwan, D., Harper, N., Slupsky, J.R. and Linley, A. (2018) Gene editing of BTK in acute myeloid leukaemia using CRISPR-Cas9.ASH
2018, San Diego, CA
96 Flynn, R., Grundmann, A., Renz, P., Hanseler, W., James, W.S., Cowley, S.A. et al. (2015) CRISPR-mediated genotypic and phenotypic correction of a
chronic granulomatous disease mutation in human iPS cells. Exp. Hematol. 43, 838–848.e3 https://doi.org/10.1016/j.exphem.2015.06.002
97 Sweeney, C.L., Choi, U., Pavel-Dinu, M., Koontz, S., Li, L., Theobald, N. et al. (2018) CRISPR-mediated targeted insertion of CYBB cDNAs into the
CYBB locus for correction of X-linked chronic granulomatous disease patient CD34+ cells.ASGCT Annual Meeting, Chicago, IL
98 Sweeney, C.L., Zou, J.Z., Choi, U., Merling, R.K., Liu, A., Bodansky, A. et al. (2017) Targeted repair of CYBB in X-CGD iPSCs requires retention of
intronic sequences for expression and functional correction. Mol. Ther. 25, 321–330 https://doi.org/10.1016/j.ymthe.2016.11.012
99 Paterna, J.C., Moccetti, T., Mura, A., Feldon, J. and Bueler, H. (2000) Influence of promoter and WHV post-transcriptional regulatory element on
AAV-mediated transgene expression in the rat brain. Gene Ther. 7, 1304–1311 https://doi.org/10.1038/sj.gt.3301221
100 Zufferey, R., Donello, J.E., Trono, D. and Hope, T.J. (1999) Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of
transgenes delivered by retroviral vectors. J. Virol. 73, 2886–2892 PMID:10074136
101 Cromer, M.K., Vaidyanathan, S., Ryan, D.E., Curry, B., Lucas, A.B., Camarena, J. et al. (2018) Global transcriptional response to CRISPR/Cas9-AAV 6- b a s e d
genome editing in CD34
+
hematopoietic stem and progenitor cells. Mol. Ther. 26,2431–2442 https://doi.org/10.1016/j.ymthe.2018.06.002
102 Haapaniemi, E., Botla, S., Persson, J., Schmierer, B. and Taipale, J. (2018) CRISPR-Cas9 genome editing induces a p53-mediated DNA damage
response. Nat. Med. 24, 927–930 https://doi.org/10.1038/s41591-018-0049-z
103 Schoggins, J.W. and Rice, C.M. (2011) Interferon-stimulated genes and their antiviral effector functions. Curr. Opin. Virol. 1, 519–525
https://doi.org/10.1016/j.coviro.2011.10.008
104 Schiroli, G., Conti, A., Ferrari, S., Della Volpe, L., Jacob, A., Albano, L. et al. (2019) Precise gene editing preserves hematopoietic stem cell function
following transient p53-mediated DNA damage response. Cell Stem Cell 24, 551–565 https://doi.org/10.1016/j.stem.2019.02.019
105 Petrillo, C., Thorne, L.G., Unali, G., Schiroli, G., Giordano, A.M.S., Piras, F. et al. (2018) Cyclosporine H overcomes innate immune restrictions to
improve lentiviral transduction and gene editing in human hematopoietic stem cells. Cell Stem Cell 23, 820–832.e9 https://doi.org/10.1016/j.stem.
2018.10.008
106 Zonari, E., Desantis, G., Petrillo, C., Boccalatte, F.E., Lidonnici, M.R., Kajaste-Rudnitski, A. et al. (2017) Efficient ex vivo engineering and expansion of
highly purified human hematopoietic stem and progenitor cell populations for gene therapy. Stem Cell Rep. 8, 977–990 https://doi.org/10.1016/j.
stemcr.2017.02.010
107 Boitano, A.E., Wang, J.A., Romeo, R., Bouchez, L.C., Parker, A.E., Sutton, S.E. et al. (2010) Aryl hydrocarbon receptor antagonists promote the
expansion of human hematopoietic stem cells. Science 329, 1345–1348 https://doi.org/10.1126/science.1191536
108 Fares, I., Chagraoui, J., Gareau, Y., Gingras, S., Ruel, R., Mayotte, N. et al. (2014) Pyrimidoindole derivatives are agonists of human hematopoietic stem
cell self-renewal. Science 345, 1509–1512 https://doi.org/10.1126/science.1256337
109 Branzei, D. and Foiani, M. (2008) Regulation of DNA repair throughout the cell cycle. Nat. Rev. Mol. Cell Biol. 9, 297–308 https://doi.org/10.1038/
nrm2351
110 Gutschner, T., Haemmerle, M., Genovese, G., Draetta, G.F. and Chin, L. (2016) Post-translational regulation of Cas9 during G1 enhances
homology-directed repair. Cell Rep. 14, 1555–1566 https://doi.org/10.1016/j.celrep.2016.01.019
© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology10
Emerging Topics in Life Sciences (2019)
https://doi.org/10.1042/ETLS20180157
111 Liu, M.J., Rehman, S., Tang, X.D., Gu, K., Fan, Q.L., Chen, D.K. et al. (2019) Methodologies for improving HDR efficiency. Front. Genet. 9, 691
https://doi.org/10.3389/fgene.2018.00691
112 Savic, N., Ringnalda, F.C.A.S., Lindsay, H., Berk, C., Bargsten, K., Li, Y.Z. et al. (2018) Covalent linkage of the DNA repair template to the CRISPR-Cas9
nuclease enhances homology-directed repair. eLife 7, e33761 https://doi.org/10.7554/eLife.33761
113 Maruyama, T., Dougan, S.K., Truttmann, M.C., Bilate, A.M., Ingram, J.R. and Ploegh, H.L. (2016) Increasing the efficiency of precise genome editing
with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat. Biotechnol. 34, 210 https://doi.org/10.1038/nbt0216-210c
114 Canny, M.D., Moatti, N., Wan, L.C.K., Fradet-Turcotte, A., Krasner, D., Mateos-Gomez, P.A. et al. (2018) Inhibition of 53BP1 favors
homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency. Nat. Biotechnol. 36,95–102 https://doi.org/10.1038/nbt.4021
115 Deau, M.C., Heurtier, L., Frange, P., Suarez, F., Bole-Feysot, C., Nitschke, P. et al. (2015) A human immunodeficiency caused by mutations in the
PIK3R1 gene. J. Clin. Invest. 125, 1764–1765 https://doi.org/10.1172/JCI81746
116 Elgizouli, M., Lowe, D.M., Speckmann, C., Schubert, D., Hulsdunker, J., Eskandarian, Z. et al. (2016) Activating PI3Kδmutations in a cohort of 669
patients with primary immunodeficiency. Clin. Exp. Immunol. 183, 221–229 https://doi.org/10.1111/cei.12706
117 Kuehn, H.S., Ouyang, W., Lo, B., Deenick, E.K., Niemela, J.E., Avery, D.T. et al. (2014) Immune dysregulation in human subjects with heterozygous
germline mutations in CTLA4. Science 345, 1623–1627 https://doi.org/10.1126/science.1255904
118 Lucas, C.L., Kuehn, H.S., Zhao, F., Niemela, J.E., Deenick, E.K., Palendira, U. et al. (2014) Dominant-activating germline mutations in the gene
encoding the PI(3)K catalytic subunit p110δresult in T cell senescence and human immunodeficiency. Nat. Immunol. 15,88–97
https://doi.org/10.1038/ni.2771
119 Schubert, D., Bode, C., Kenefeck, R., Hou, T.Z., Wing, J.B., Kennedy, A. et al. (2014) Autosomal dominant immune dysregulation syndrome in humans
with CTLA4 mutations. Nat. Med. 20, 1410–1416 https://doi.org/10.1038/nm.3746
© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology 11
Emerging Topics in Life Sciences (2019)
https://doi.org/10.1042/ETLS20180157