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REVIEW
published: 15 March 2021
doi: 10.3389/fgeed.2021.630600
Frontiers in Genome Editing | www.frontiersin.org 1March 2021 | Volume 3 | Article 630600
Edited by:
Vania Broccoli,
San Raffaele Hospital (IRCCS), Italy
Reviewed by:
Josep M. Canals,
University of Barcelona, Spain
Lorenzo A. Cingolani,
Italian Institute of Technology (IIT), Italy
*Correspondence:
Manju A. Kurian
manju.kurian@ucl.ac.uk
†These authors share first authorship
‡These authors share last authorship
Specialty section:
This article was submitted to
Genome Engineering and Neurologic
Disorders,
a section of the journal
Frontiers in Genome Editing
Received: 17 November 2020
Accepted: 19 February 2021
Published: 15 March 2021
Citation:
McTague A, Rossignoli G, Ferrini A,
Barral S and Kurian MA (2021)
Genome Editing in iPSC-Based Neural
Systems: From Disease Models to
Future Therapeutic Strategies.
Front. Genome Ed. 3:630600.
doi: 10.3389/fgeed.2021.630600
Genome Editing in iPSC-Based
Neural Systems: From Disease
Models to Future Therapeutic
Strategies
Amy McTague 1,2† , Giada Rossignoli 1†, Arianna Ferrini 1, Serena Barral 1‡ and
Manju A. Kurian 1,2
*‡
1Developmental Neurosciences, Great Ormond Street Institute of Child Health, University College London, London,
United Kingdom, 2Department of Neurology, Great Ormond Street Hospital, London, United Kingdom
Therapeutic advances for neurological disorders are challenging due to limited
accessibility of the human central nervous system and incomplete understanding of
disease mechanisms. Many neurological diseases lack precision treatments, leading to
significant disease burden and poor outcome for affected patients. Induced pluripotent
stem cell (iPSC) technology provides human neuronal cells that facilitate disease
modeling and development of therapies. The use of genome editing, in particular
CRISPR-Cas9 technology, has extended the potential of iPSCs, generating new models
for a number of disorders, including Alzheimers and Parkinson Disease. Editing of iPSCs,
in particular with CRISPR-Cas9, allows generation of isogenic pairs, which differ only in
the disease-causing mutation and share the same genetic background, for assessment
of phenotypic differences and downstream effects. Moreover, genome-wide CRISPR
screens allow high-throughput interrogation for genetic modifiers in neuronal phenotypes,
leading to discovery of novel pathways, and identification of new therapeutic targets.
CRISPR-Cas9 has now evolved beyond altering gene expression. Indeed, fusion of a
defective Cas9 (dCas9) nuclease with transcriptional repressors or activation domains
allows down-regulation or activation of gene expression (CRISPR interference, CRISPRi;
CRISPR activation, CRISPRa). These new tools will improve disease modeling and
facilitate CRISPR and cell-based therapies, as seen for epilepsy and Duchenne muscular
dystrophy. Genome engineering holds huge promise for the future understanding and
treatment of neurological disorders, but there are numerous barriers to overcome. The
synergy of iPSC-based model systems and gene editing will play a vital role in the route
to precision medicine and the clinical translation of genome editing-based therapies.
Keywords: induced pluripotent stem cells, CRISPR, gene editing, precision treatment, CRISPRi, CRISPRa,
neurological disorders, disease modeling
McTague et al. Genome Editing in iPSC Neural Systems
INTRODUCTION
Neurological disorders are the leading cause of disability and
the second leading cause of death worldwide (Feigin et al.,
2017). Despite apparent significant advances in investigation and
treatment of these diseases, the health burden of neurological
disorders has risen since 1990 (Feigin et al., 2019). In addition,
the economic and societal impact of neurological disorders is
substantial. As a result of the significant morbidity of many
neurological conditions, the estimated annual cost of the nine
commonest neurological disorders to society in the USA was
$0.8 trillion (Gooch et al., 2017). A similar study in Europe
found the cost of the 19 most prevalent brain and mental health
disorders to be e798 billion per year (Olesen et al., 2012).
Neurological disorders also include many rare diseases that are
associated with considerable morbidity, societal, and economic
impact (Graf von der Schulenburg and Frank, 2015). One of
the reasons for this significant healthcare burden is that many
neurological disorders still lack effective targeted treatments. The
simultaneous development of two ground-breaking technologies,
induced pluripotent stem cells (iPSCs) and genome engineering,
has the potential to change this.
Development of effective treatments for neurological
disorders is often challenging. The near inaccessibility of the
human central nervous system hampers the investigation of
underlying disease mechanisms and ascertainment of therapeutic
targets. Animal models and heterologous cellular in vitro systems
have provided insights into pathophysiological pathways of
several neurological disorders, leading to advances in therapeutic
approaches. Nevertheless, these models do not fully mimic
human physiology, metabolism, and homeostasis, and only
partially recapitulate the progression of the disease (Barral and
Kurian, 2016). As a result, there is a higher failure rate of clinical
trials and novel therapy development for neurological diseases
(Kinch, 2015).
DERIVING NEURONAL SYSTEMS FROM
iPSC
With the advent of pluripotent stem cell-based technologies,
both embryonic stem cells (ESCs) (Thomson, 1998) and induced
pluripotent stem cells (iPSCs) (Takahashi et al., 2007), a new
source of human neural cells became available. In particular,
patient-derived iPSCs are enabling insight into rare neurological
disorders for which no suitable models are available and
are an ideal platform to test therapeutic approaches (Barral
and Kurian, 2016; Mertens et al., 2016; Ghaffari et al.,
2018; Kampmann, 2020; Silva and Haggarty, 2020). To date,
several protocols have been published for the differentiation of
pluripotent stem cells into neural cellular subpopulations, among
which cortical, motor, dopaminergic, GABAergic, cholinergic,
serotonergic neurons (Wada et al., 2009; Erceg et al., 2010;
Wicklund et al., 2010; Kriks et al., 2011; Juopperi et al.,
2012; Kirkeby et al., 2012; Carri et al., 2013; Espuny-Camacho
et al., 2013; Liu et al., 2013; Maroof et al., 2013; Nicholas
et al., 2013; Zhang et al., 2014; Du et al., 2015; Nizzardo
et al., 2015; Hu et al., 2016; Lu et al., 2016; Nolbrant et al.,
2017), and microglial cells, astrocytes and oligodendrocytes
(Nistor et al., 2005; Ogawa et al., 2011; Emdad et al., 2012;
Juopperi et al., 2012; Wang S. et al., 2013; Douvaras et al.,
2014; Muffat et al., 2016; Abud et al., 2017; Tcw et al., 2017).
The majority of these protocols are able to reproduce in a
two-dimensional (2D) cellular model some of the processes
observed in central nervous system (CNS) development and
to derive neural populations that resemble those observed in
the human fetal period. In order to overcome the lack of
tissue-like complexity and the early stage fetal nature of the
derived neural cells present in 2D derived neuronal systems,
new protocols have been developed for the generation of
three-dimensional (3D) brain organoids, which recapitulate
some aspects of late gestational human brain (Lancaster et al.,
2013; Pasca, 2018, 2019). Brain organoids not only allow
complex neuronal network formation but can be maintained
for long periods in vitro allowing analysis of neural circuit
formation and complex physiological mechanisms which align
more closely with human brain physiology in normal and
disease states (Quadrato et al., 2017; Trujillo et al., 2019).
However, maintaining cerebral organoids in long-term culture
remains challenging due to oxygen, nutrient supply, and micro-
environment issues, largely related to the inherent lack of
vasculature. This was illustrated by intracerebral engraftment
of human iPSC-derived cerebral organoids in mice, which
led to vascularization, functional connectivity, and improved
survival of organoids (Mansour et al., 2018). Giandomenico
et al. used an air:liquid interface with sliced cerebral organoids
to allow perfusion of the organoid core, improving neuronal
survival, maturation, and axonal outgrowth and resulting in
a functional circuit with mouse spinal cord explants in co-
culture (Giandomenico et al., 2019, 2020). Other strategies to
vascularize cerebral organoids have included ectopic expression
of ETV2 (Cakir et al., 2019) or co-culture with iPSC-derived
endothelial cells (Pham et al., 2018) or human umbilical vein
endothelial cells (Shi et al., 2020). Advances in bioengineering
and biomaterials have also sought to better reproduce the native
extracellular matrix microenvironment. Although Matrigel and
hydrogels are most commonly used, a floating scaffold consisting
of synthetic microfilaments produced elongated embryoid bodies
and improved cortical development in organoids(Lancaster et al.,
2017). Bioreactors have also been developed to allow perfusion
of nutrients and oxygen to organoid tissues (Lancaster et al.,
2013; Qian et al., 2016). At the same time as improvements in
organoid culture systems and subsequent to the development of
whole-brain organoids, protocols using growth factor or small
molecule regional patterning emerged, generating forebrain
(Pasca et al., 2015; Birey et al., 2017; Quadrato et al., 2017;
Sloan et al., 2018), midbrain-like (Jo et al., 2016; Qian et al.,
2016), and hindbrain/cerebellar organoids (Muguruma, 2018) in
addition to specific regional structures such as hypothalamic/
pituitary and retinal organoids (Parfitt et al., 2016; Kasai et al.,
2020). These advances were followed by fusion of regional
organoids to create subpallial-cortical fused organoids (Bagley
et al., 2017; Birey et al., 2017; Xiang et al., 2017; Marton
and Pa¸sca, 2020), allowing the study of interactions between
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McTague et al. Genome Editing in iPSC Neural Systems
medial ganglionic eminence-derived GABAergic neurons and
forebrain glutamatergic neurons. Most recently, cortico-striatal
assembloids with functional neural circuits (Miura et al.,
2020) and cortico-motor assembloids of hindbrain, spinal cord,
and muscle have been generated (Andersen et al., 2020),
demonstrating the potential of these models to investigate multi-
organ aspects of neurological diseases.
iPSCs patient-derived neurons, therefore, represent a unique
in vitro model for the study of neurological disorders. They are an
unlimited source of patient-derived cells, which retain the genetic
background of the donor and allow investigation of disease-
related mechanisms. Therefore, they provide a humanized
model in which to test novel treatments, with the potential
to accelerate the translation of novel therapeutic approaches.
Moreover, they provide a renewable source of cells for cell
replacement strategies to treat neurodegenerative disorders
(Figure 1).
Since their inception, gene editing technologies have
proved a useful tool in the development of in vitro disease
models. Replacement of endogenous genomic sequences
with exogenous donor DNA via homologous recombination
(HR) and correct insertion of exogenous DNA at defined
mammalian chromosomal locations was first developed in the
1980s (Smithies et al., 1984), and subsequently applied to the
genomic modification of mouse embryonic stem cells (ESCs)
(Hasty et al., 1991). The discovery of I-SceI yeast meganuclease
(Jacquier and Dujon, 1985), which promotes HR endogenous
cellular mechanisms to repair DNA double-strand breaks (DSBs)
in the presence of donor DNA, lead to the establishment of
genome-editing strategies in murine cells (Choulika et al., 1995)
and ESCs (Cohen-Tannoudji et al., 1998) based on proteins
derived from unicellular organisms.
The advent of DNA-binding zinc-finger nuclease (ZFN)
technology improved efficiency in genome-editing of
mammalian cells (Bibikova et al., 2001), leading to the generation
of the first knockout rat (Geurts et al., 2009). Following use in
animals and cellular models (Petersen and Niemann, 2015),
ZFNs-based genome editing was exploited for the correction of
genetic mutations in patient-derived iPSCs (Soldner et al., 2011;
Reinhardt et al., 2013; Kiskinis et al., 2018; Wang et al., 2018;
Korecka et al., 2019), or for insertion of known disease-relevant
mutations in iPSCs derived from healthy individuals (Verheyen
et al., 2018), allowing direct investigation of specific genomic
alterations and disease phenotypes. In addition, ZFNs were
applied for the generation of engineered lines to study cell fate
determination and improve iPSCs differentiation protocols
(Hockemeyer et al., 2009), as well as to produce cell type-specific
reporter systems for the investigation of disease pathogenesis
(Zhang et al., 2016).
Genome editing technology further advanced with the advent
of transcription activator-like effector nucleases (TALENs),
which proved to be an efficient technology for the generation
of animal models (Tesson et al., 2011). TALENs were further
employed in the study of neurological disorders through the
introduction of disease-causing mutations in control iPSCs
(Wen et al., 2014; Lenzi et al., 2015; Akiyama et al., 2019)
and/or correction of genetic mutations in patient-derived iPSCs
(Maetzel et al., 2014; Wen et al., 2014; Li H. L. et al., 2015; Tanaka
et al., 2018; Akiyama et al., 2019) leading to greater confidence in
disease-underlying mechanisms and development of therapeutic
approaches. Moreover, TALENs technology was used to develop
reporter lines for stem cell-based research (Cerbini et al., 2015;
Pei et al., 2015).
Rapidly following the development of TALENs technology,
clustered regularly interspaced short palindromic repeats
(CRISPR) with the CRISPR-associated protein (Cas9) system
(Gasiunas et al., 2012; Jinek et al., 2012) demonstrated
revolutionary potential to engineer the genome of mammalian
cells in culture (Cong et al., 2013; Mali et al., 2013) and animal
models (Wang H. et al., 2013). As for ZFNs or TALENs, CRISPR-
Cas9 uses distinct DNA cleavage and binding modules. However,
CRISPR-Cas9 system uses its own natural endonuclease and
relies on a CRISPR RNA (crRNA) and a trans-activating RNA
(transRNA) to specifically bind target DNA sequences and
activating Cas9. Therefore, the long and complex process of
engineered nuclease production was rapidly overcome by the
plasticity and simplicity of generating different CRISPR-based
approaches, which require only the design of a specific target-
matching RNA. The extraordinary efficacy of CRISPR-Cas9,
together with its great versatility for the generation of a broad
range of substitutions, duplications, deletions, inversions,
and many other complex alterations up to chromosomal
rearrangements, have transformed the genome-editing field.
There were, however, several limitations that required further
improvements. Increased efficiency and reduction of off-target
effects have been achieved through the engineering of Cas9
protein (Kleinstiver et al., 2015, 2016; Anders et al., 2016;
Slaymaker et al., 2016; Chen et al., 2017; Casini et al., 2018;
Hu et al., 2018; Lee et al., 2018; Nishimasu et al., 2018) and
amendments to the design and structure of the guide RNA
(Jinek et al., 2012; Hsu et al., 2013; Cui et al., 2018; Filippova
et al., 2019; Moon et al., 2019), as well as the discovery and
application of Cas proteins with different and specific gene-
editing properties (Zetsche et al., 2015; Abudayyeh et al.,
2016; Burstein et al., 2017). CRISPR-based technology has
further developed to allow transcriptional inhibition (CRISPR
interference, CRISPRi) or activation (CRISPR activation,
CRISPRa). This CRISPR-based transcriptional modulation is
achieved by repressor or activator transcription domains fused
to a catalytically inactive Cas9 (dCas9) and guide RNAs directed
to the promoter or regulatory regions of specific genes (Gilbert
et al., 2013).
Owing to its robustness and flexibility, CRISPR-based gene
editing systems have proven efficient for gene modification, gene
expression regulation, epigenetic modulation, and chromatin
manipulation, at both single-gene level and large-scale screening
(Adli, 2018). Therefore, CRISPR-based technology has quickly
become the preferred method of choice for genome-editing,
particularly in iPSC model systems.
In this review, we will examine the role of genome engineering
in iPSC-derived models of neurological disorders and discuss
how the combination of these technologies has the potential
to advance our understanding of disease mechanisms and to
accelerate the translation of gene editing therapies.
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McTague et al. Genome Editing in iPSC Neural Systems
FIGURE 1 | Use of patient-derived Induced Pluripotent Stem Cells (iPSC) for neurological disorders. (Left) iPSCs are readily derived from patients and controls
through cellular reprogramming. Patient- and control-derived iPSCs are differentiated into the neuronal type relevant to the specific disease. Gene editing techniques
such as CRISPR-Cas9 can be utilized to genetically correct the mutation in iPSCs to obtain isogenic control lines which only differ from the mutated iPSC lines by the
genetic variant of interest. (Middle) Following genome editing and differentiation of iPSCs, derived cells are used to gain phenotypic insights into the specific disease
mechanism through a variety of functional, morphological, and molecular analysis. (Right) Once a disease phenotype has been identified, iPSC-derived neuronal cells
can be utilized for drug screens, which could accelerate personalized therapies for neurological disorders.
Creating New Disease Models for
Neurological Disorders
The parallel development of iPSC and gene editing technologies
has been serendipitous for advancing the understanding of
neurological disorders (Figure 2) (Hockemeyer and Jaenisch,
2017). The ability to model neurological disorders in a
humanized patient-derived model system has already led to
many insights into disease mechanisms (Figure 1) (Barral and
Kurian, 2016; Li et al., 2018), and this has been accelerated by
synergy with genome editing. Initially TALENs and subsequently
CRISPR-Cas9, have been utilized to create point mutations,
insertion-deletions and to edit repeat expansions in iPSC lines
from healthy control subjects (Supplementary Table 1). This has
been particularly helpful in rare diseases where there is reduced
accessibility to patient-derived samples for the generation of
patient-derived iPSCs or where there is difficulty in obtaining
patient samples with a particular genotype, such as CHD8-related
autistic spectrum disorder (ASD) (Wang P. et al., 2017) and
KCNT1-related epilepsy (Quraishi et al., 2019). Gene editing of
patient-derived cells in rare neurological diseases such as spinal
muscular atrophy led to phenotypic rescue with restoration
of SMN expression, motoneuron survival, and neuromuscular
junction formation and has paved the way for novel therapeutic
approaches (Zhou et al., 2018). In Huntington’s disease (HD),
it has long been observed that CAG repeat length correlates
with the age of disease onset. In human embryonic stem cells
edited to contain differing CAG repeat lengths, cell-type specific
phenotypic differences were seen which related to CAG length
(Ooi et al., 2019). In addition, genome editing of control lines
allows the study of many variants at once in the same genetic
background, which may be more practical than collecting large
numbers of patient lines. This paradigm has been elegantly
used by Kwart et al., who generated a panel of 15 different
familial Alzheimer disease (AD) variants in two genes PSEN1
and APP to look for common disease mechanisms (Kwart
et al., 2019). Using transcriptomic analysis, they were able to
identify accumulation of βCTF mediating a common endosomal
dysfunction (Supplementary Table 1).
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McTague et al. Genome Editing in iPSC Neural Systems
FIGURE 2 | Timeline showing the parallel development of iPSC and gene editing technologies which have advanced in vitro modeling of neurological diseases. The
combination of these technologies has the potential to advance our understanding of disease mechanisms and to accelerate the translation of gene editing therapies
into the clinic.
Isogenic Controls Reduce Variability and
Improve Modeling
One of the strengths of the iPSC technology paradigm is the
ability to model the effects of a variant in the native genetic
milieu, which will naturally differ between individuals as part
of normal genetic variation. Whilst iPSC systems offer an
unparalleled opportunity to study and annotate human genetic
variants associated with disease, this also represents a major
shortcoming. Experimental variability due to reprogramming or
differentiation differences between lines also make a contribution
to system noise. In earlier iPSC studies, unrelated controls,
often from in-house or stem cell bank lines or unaffected family
members, were used for comparison with patient-derived mutant
lines. However, extensive transcriptomic analyses from large stem
cell repositories such as Human Induced Pluripotent Stem Cell
Initiative (HipSci), European Bank for Induced pluripotent Stem
Cells (EBiSC), and National Heart, Lung, and Blood Institute
(NHBLI) have shown that variance is most greatly influenced
by genetic differences between individuals (Germain and Testa,
2017) rather than between technical replicates, such as different
clones from a single patient.
Therefore, for robust comparison of phenotypic effects, an
isogenic line, which only differs by the genetic variant of
interest, is now deemed necessary to ensure that observed
differences are attributable to a specific genetic defect. The advent
of gene editing technologies has revolutionized this aspect of
iPSC modeling and has led to a great number of phenotypic
insights (Supplementary Table 1). For example, early iPSC
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McTague et al. Genome Editing in iPSC Neural Systems
models of SCN1A-related epilepsies suggested conflicting disease
mechanisms, with some studies finding increased sodium current
in excitatory neurons contradicting the loss of function noted
in GABAergic neurons in animals and other iPSC studies.
Liu et al. used TALENs to correct an SCN1A variant from a
patient-derived line and CRISPR-Cas9 to knock in a fluorescent
reporter expressed in GABAergic neurons (Liu et al., 2016).
By comparison with isogenic controls, they were able to show
a selective loss of function in GABAergic neurons, which was
later confirmed in another study (Sun et al., 2016). Subtler
phenotypes in later-onset disorders such as Parkinson’s disease
(PD) have also been revealed by correction of disease-causing
variants; Reinhardt et al. demonstrated that cellular phenotypes
such as neurite outgrowth, abnormal autophagy, and tau and
α-synuclein accumulation were LRRK2-dependent in familial
PD (Reinhardt et al., 2013). Isogenic controls also have the
power to show that some cellular phenotypes are dependent on
genetic background even in monogenetic conditions; correction
of LRRK2 variants in another study exposed both LRRK2-
dependent and LRRK2-independent effects, which were likely
related to genetic background and reflected the heterogeneous
clinical features and variable severity in familial PD (Nickels
et al., 2019). This enabled the identification of serine metabolism
as a putative genetic modifier of familial PD. Isogenic controls
are therefore essential in both monogenic highly penetrant
disorders and disorders where there is phenotypic variability.
For example, in neurological diseases caused by triplet repeat
expansions such as Friedrich ataxia (FA) there is significant
clinical heterogeneity. This can be due to both gene-dependent
factors such as expansion size and gene-independent factors
such as genetic background and environmental factors (Schreiber
et al., 2019). Therefore, isogenic controls were necessary to
discriminate truly FRDA-intrinsic effects, with restoration of
frataxin expression and other FA phenotypic features in response
to ZFN excision of the expanded GAA repeat (Li Y. et al., 2015).
Gene Editing to Investigate Complex
Disorders
It would be expected that genetic modifiers make an even larger
contribution to complex diseases, along with environmental
and non-genetic risk factors such as lifestyle. Genome-wide
association studies (GWAS) identify risk loci which are
statistically associated with the risk of developing complex
disorders such as sporadic PD or AD, but the single nucleotide
polymorphisms or alleles identified usually have individually
small effect sizes, making detection of a subtle phenotype in
an iPSC system challenging. Gene editing can answer some of
these challenges. Differing genotypes of the apolipoprotein E4
gene APOE4 are associated with risk of sporadic AD (Raman
et al., 2020). iPSCs from healthy subjects were converted from
neutral risk (APOE3) to APOE4 (high risk) by gene editing, and
patient-derived iPSCs were conversely converted from APOE4
to APOE3 in several studies (Lin et al., 2018; Meyer et al.,
2019). This “rescue” of the risk status of iPSCs derived from
patients with the propensity to develop AD later in life led to a
reversal of AD phenotypes such as the inability of glial cells to
clear extracellular Aβand increased Aβaggregates in cerebral
organoids. In addition, accelerated neuronal differentiation and
reduced renewal of neural progenitors were reversed by gene
editing (Meyer et al., 2019). This study also highlighted that iPSC
systems could be highly useful to investigate late-onset disorders,
which may seem counter-intuitive as iPSC-derived neurons are
considered immature, representing a fetal neuronal development
stage even after several months in culture (Vera and Studer,
2015). Nevertheless, iPSC systems represent an opportunity to
model early neurodevelopment and therefore may detect early
disease features which are pre-symptomatic, such as the APOE4-
dependent altered neuronal differentiation seen in the sporadic
AD model (Meyer et al., 2019), which could result in neuronal
vulnerability to other stressors and environmental factors.
Gene editing can also be used to investigate the cellular effects
of risk alleles in models of sporadic disease. Schrode et al. used
CRISPR-Cas9 to introduce a known risk allele into control iPSC
lines and CRISPRi/a to modulate the top-ranked schizophrenia-
associated expressive quantitative trait loci (eQTL) (Schrode
et al., 2019), including SNAP91,TSNARE1, and CLCN3. An
eQTL is a genomic region or single nucleotide polymorphism
(SNP) associated with the expression of a gene, in this case,
identified from post-mortem (PM) gene expression studies in
schizophrenia patients. Extensive phenotypic analyses of neurons
differentiated from isogenic pairs showed that although there
were gene-dependent effects, these converged on a common
synaptic defect. There was a particular phenotypic overlap
between the effects of SNAP91 CRISPRi and TSNARE1 CRISPRa.
In addition, this study showed that the effect sizes seen in the
iPSC-derived neurons and organoids were larger than those
in PM studies. Moreover, the combinatorial perturbation of
genes rather than an additive model better approximated gene
expression signatures in schizophrenia datasets and revealed
enrichment in gene sets involved in secretion of glutamate
and other neurotransmitters, synaptic vesicle trafficking, and a
postsynaptic glutamate receptor pathway. This demonstration of
a complex synergistic impact of common variants on cellular
and molecular phenotypes for schizophrenia emphasizes the
importance of considering this particular disorder’s polygenic
nature and may apply more broadly to many complex non-
Mendelian genetic disorders. Potentially, in vitro systems such
as iPSC models could be used to identify novel eQTLs such as
SNPs, which modify gene expression in different iPSC-derived
cell lineages, and which can then be directly investigated for their
functional effect (Soldner and Jaenisch, 2019).
Interrogating the Transcriptome and
Methylome With Genome Editing
Genome-wide analysis of gene expression in iPSC models using
RNA sequencing has been made possible by the generation of
appropriate isogenic controls and has led to many mechanistic
insights (Supplementary Table 1). CHD8 has been found to be
mutated in both ASD and schizophrenia. To understand the
downstream effects of CHD8 variants and identify common
pathways, Wang et al. generated CHD8 knockout lines using gene
editing (Wang P. et al., 2017). RNAseq revealed CHD8 regulates
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McTague et al. Genome Editing in iPSC Neural Systems
known ASD genes TCF4 and AUTS2 and affects GABAergic
interneuron development by modulating DLX gene expression.
Pathway analysis of differentially expressed genes (DEGs) was
enriched for genes involved in the Wnt/β-catenin pathway,
representing a potential drug target.
Gene editing in iPSC systems has been instrumental in
understanding pathophysiological mechanisms related to the
modification of a disease-associated gene. However, genome
engineering can be used in combination with transcriptomic
analysis to better investigate underlying disease mechanisms. To
investigate early-onset AD in patients with Down syndrome,
CRISPR-Cas9 was used to delete the supernumerary copy of
APP in T21 lines and inducible CRISPRa to upregulate APP
gene expression (Ovchinnikov et al., 2018). APP gene dosage
was shown to be connected to β-amyloid production but not
to other AD cellular phenotypes, including apoptosis. The use
of CRISPR screens to delineate disease mechanisms is discussed
further below.
In addition to DNA editing, it is possible to alter DNA
methylation states using similar genome engineering approaches.
Proof of principle was established with gene editing in Fragile
X syndrome, a common inherited form of intellectual disability
caused by expansion of a CGG repeat in the FMR1 gene. CRISPR-
Cas9 editing of the repeats revealed their role in demethylation of
the upstream CpG island of the FMR1 promoter, resulting in an
open chromatin state and initiation of transcription with FMR1
levels restored in derived neurons (Park et al., 2015). A further
study used a catalytically inactive Cas9 (dCas9) fused to a DNA
methyltransferase domain (dCas9-Tet1) to directly demethylate
the CGG repeat, rescuing FMR1 expression (Liu et al., 2018). A
similar approach was taken by Kantor et al. to demethylate SNCA
in a PD model, rescuing SNCA levels and mitophagy defects in
patient-derived dopaminergic neurons without altering the rest
of the methylome (Kantor et al., 2018).
Modeling Neurodevelopmental Impact,
Cell-Type Vulnerability, and Multi-System
Diseases
A further advantage of genome engineering in iPSC systems
is the ability to assess the impact of gene dosage and
genetic variation at differing neurodevelopmental time-points.
In tuberous sclerosis, heterozygous variants in TSC1 or TSC2
(mTOR pathway genes) cause a multisystem disorder including
epilepsy, developmental delay and propensity to non-malignant
overgrowth such as cortical tubers. Martin et al. examined the
impact of tuberous sclerosis variants on neuronal precursors,
noting that MTOR inhibitors such as rapamycin did not restore
abnormal proliferation and neurite outgrowth to the same
degree as gene correction, indicating mTOR-independent early
disease mechanisms that have important implications for future
treatments (Martin et al., 2020). Previous studies have identified
biallelic TSC1/2 mutations in cortical tubers, suggesting a second
hit or somatic mutation may be required (Winden et al., 2019).
Indeed TALEN or CRISPR-engineered biallelic patient lines
showed an effect of gene dosage on mTOR activation and reduced
synaptic activity (Sundberg et al., 2018; Winden et al., 2019). Blair
et al. edited a control iPSC line to create a TSC2 loss of function
variant with an inducible loss of function variant in the other
allele (Blair et al., 2018). During expansion of neural progenitors,
mosaic biallelic inactivation was shown to lead to formation
of dysplastic cells, supporting the two-hit hypothesis. Indeed,
inducible gene editing in cerebral organoids could provide a
viable future platform to investigate disease mechanisms in other
similar disorders where cerebral somatic mosaicism is implicated,
such as malformations of cortical development (Poduri et al.,
2012; Verheijen, 2019).
iPSC modeling is ideal for disorders such as tuberous sclerosis
due to the early onset of clinical disease and the ability
to generate relevant neuronal types of any lineage, such as
cerebellar Purkinje cells or neural crest cells (Sundberg et al.,
2018; Delaney et al., 2020). Cell-type vulnerability can also be
investigated with iPSC models and gene editing. In AD, some
brain regions appear to be more severely affected by Aβplaque
deposition. Neuronal differentiation was directed to either to
caudal (hindbrain) or rostral (forebrain) fates from patient-
derived iPSCs bearing APP mutation and showed more severe
tau responses in forebrain neurons, confirming brain-region
susceptibility in AD (Muratore et al., 2017). Investigation of the
impact of the APOE4 genotype in microglia also revealed relative
cell-type vulnerability for sporadic vs. familial AD (Konttinen
et al., 2019).
Beyond the central nervous system, the ability to generate
multiple lineages is helpful for multi-system disorders. In
SCN1A-Dravet syndrome, increased risk of SUDEP may relate
to cardiac sodium channel dysfunction (Frasier et al., 2018).
Cardiomyocytes were generated from patient-derived iPSCs;
mutant cardiomyocytes showed increased sodium current
density and spontaneous contraction rates, partly explained by
a compensatory increase in SCN5A expression. In Nieman-Pick
disease, both neurons and hepatocytes were differentiated from
patient-derived iPSCs with biallelic mutations in NPC1 (Maetzel
et al., 2014). Phenotypic rescue of the autophagy defect was
demonstrated by TALEN gene correction in both cell lineages
with implications for future therapy. In FA, cardiomyopathy
is a major cause of morbidity and mortality. ZFN editing of
patient-derived iPSC and control lines to create isogenic controls
showed rescue of frataxin expression, lipid accumulation, and a
hypertrophy-specific transcriptomic signature (Li J. et al., 2019).
Most recently, iPSCs from FA patients were differentiated into
dorsal root ganglia organoids, generating sensory neurons, which
were co-cultured with intrafusal muscle fibers, recapitulating
the human proprioceptive network (Mazzara et al., 2020). This
enabled testing of two different CRISPR therapeutic approaches,
with improved phenotypic rescue following excision of the entire
first intron rather than the expanded GAA tract alone.
Exploiting the pluripotency of iPSCs can also reveal tissue-
specific putative disease mechanisms and treatment approaches.
Oikari et al. investigated the impact of familial AD mutations in
PSEN1 on blood brain barrier (BBB) formation by differentiating
induced brain endothelial cells (iBECs) from patient-derived
and isogenic lines (Oikari et al., 2020). Mutant iBECs showed
abnormal tight and adherin junction protein expression. Focused
ultrasound is a novel approach to open the BBB. In iBEC cultures,
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AD and isogenic iBECs responded differently, suggesting this
may be a novel approach to improve CNS drug delivery in AD.
Combining Genome Editing With
Environmental Perturbation
It is also possible to combine iPSC systems and genome
engineering to study the additional effect of environmental
factors or stressors. Both mutations in the tau gene, MAPT,
and exposure to environmental toxins such as heavy metals
are known to increase the risk of developing progressive
supranuclear palsy, a neurodegenerative disorder characterized
by the accumulation of tau aggregates. Induced neurons
differentiated from a patient-derived MAPT iPSC line and the
corresponding gene-corrected isogenic control were exposed to
heavy metals, including chromium and nickel (Alquezar et al.,
2020). Heavy metals increased tau levels and induced apoptosis
in all cell lines, but MAPT mutant lines showed increased
vulnerability, indicating a link between genetic predisposition
and environmental factors. Environmental factors in the form
of temperature can worsen some genetic conditions. Pathogenic
variants in ATP1A3 cause a range of neurological conditions,
including alternating hemiplegia of childhood, where attacks of
hemiplegia can be triggered by stressors such as illness or change
in temperature. Patient-derived neurons displayed hyperactivity
on microelectrode array analysis following temperature elevation
compared to gene-corrected neurons (Snow et al., 2020).
Similarly, ultra-violet irradiation of iPSC-derived neuronal
cultures from a patient with ERCC6-related Cockayne syndrome
recapitulated the DNA damage and premature aging seen in vivo,
which were rescued in the gene-corrected lines (Wang et al.,
2020).
CRISPR-CAS9 BASED SCREENING FOR
NEUROLOGICAL DISORDERS
CRISPR Knock-Out and Knock-In
Screening for Disease-Specific
Investigation
In addition to numerous applications in single-gene engineering,
CRISPR-Cas9 has evolved into a functional genomics tool.
It has been adapted to target multiple genes simultaneously,
giving rise to several genome-wide CRISPR knock-out (CRISPR
KO) and knock-in (CRISPR KI) studies (Cong et al., 2013).
CRISPR-Cas9 based screening combined with high throughput
methods for functional evaluation is a powerful approach to
systematically elucidate gene function in health and disease
states, with further implications for diagnosis and treatment
development (Figure 3).
Genome-wide CRISPR-Cas9 screens were initially employed
in dividing immortalized cell-lines, allowing the reliable
introduction of variants at known sites in the genome,
facilitating selection of clones, and easy scaling-up of functional
evaluation. This “forwards genetics” approach allowed unbiased
phenotypic screening and identified genetic determinants
of diptheria infection and melanoma treatment resistance,
amongst other examples (Shalem et al., 2014; Wang et al., 2014).
However, heterologous cellular models lack relevant cellular
pathophysiological phenotypes, in particular for neurological
disorders. Therefore, patient iPSC-derived neuronal cells
have been employed as a new in vitro model for secondary
validation of identified hits. Potting and colleagues (Potting
et al., 2017) performed a selection screen in HEK293 cells
expressing endogenous PARKIN, an E3 ligase that promotes
mitophagy by ubiquitinating mitochondrial proteins and whose
mutations cause familial PD. Among 53 identified positive and
negative regulators of PARKIN expression, a transcriptional
repressor network including THAP11 was further validated in
iPSCs-derived excitatory neurons. Heterozygous KO of THAP11
significantly increased PARK2 mRNA and PARKIN protein
levels and altered mitochondrial ubiquitinylation in neurons.
This analysis demonstrated that PARKIN levels affect mitophagy
in the early damage-induced state, further elucidating the role of
mitophagy in PD’s pathogenesis.
Similarly, a CRISPR KO screen was performed to understand
underlying mechanisms and therapeutic targets for treatment
of frontotemporal dementia and amyotrophic lateral sclerosis
(c9FTD/ALS) (Kramer et al., 2018). Both neurological conditions
are characterized by hexanucleotide repeat expansions in
C9orf72 gene, which translate into dipeptide repeat (DPR)
aggregation-prone proteins. CRISPR-Cas9 screening was initially
performed in myelogenous leukemia cells to identify suppressors
and enhancers of C9orf72 DPR-mediated toxicity. A second
validation was then performed in primary mouse neurons,
and the effect of a final hit was unraveled in motor neurons
derived from C9orf72-ALS iPSCs. This approach identified
endoplasmic reticulum (ER) function and stress as important
mechanisms involved in c9FTD/ALS pathogenesis. In addition,
it allowed the identification of novel mitigators of DPR-induced
toxicity, such as down-regulation of the ER-resident protein
TMX2, which improved ALS-derived motor neurons survival
and could represent a potential therapeutic target. Another
CRISPR KO screen was performed in retinal pigment epithelium
cells with validation in patient-derived iPSCs and differentiated
motor neurons to discover genetic modifiers of C9ORF72 DPR
production (Cheng et al., 2019). Among enriched genes involved
in RNA nuclear export and translation, DDX3X, an RNA
helicase, was identified to directly bind the repeat expansions
in C9orf72 RNA, repressing the translation of DPR proteins.
Decreasing DDX3X increased endogenous DPRs in patients’
iPSCs, while exogenous expression of DDX3X reduces DPR
levels and rescues nucleocytoplasmic transport abnormalities,
and improved survival of iPSC-differentiated motor neurons.
This work indicate that strategies to increase DDX3X expression
or activity may have therapeutic potential for c9FTD/ALS.
To date, very few genome-wide CRISPR KO and CRISPR
KI screens have been undertaken for the study of neurological
disorders, and just a small fraction was directly performed
on iPSC-derived neurons. This is likely a consequence of the
considerable resources and investments in time that iPSC-
based systems require. This issue has been addressed with
two approaches: (a) perform genome-wide screening in iPSCs
or iPSCs-derived cellular subtypes generated from healthy
individuals, which can be followed by hit validation in a disease
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McTague et al. Genome Editing in iPSC Neural Systems
FIGURE 3 | CRISPR-Cas9 for gene editing and gene expression modulation. Genome engineering using CRISPR-Cas9 can be used for gene editing with insertions
or deletions resulting in gene knock-out, or for knock-in using homologous recombination. Recently, CRISPR has evolved toward gene expression modulation;
CRISPR intereference (CRISPRi) and CRISPR activation (CRISPRa) can be used for transient inhibition or activation of gene expression respectively.
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McTague et al. Genome Editing in iPSC Neural Systems
model; (b) perform a focused analysis on a wide set of genes or
mutations known to confer susceptibility to a particular disease.
The first approach is exemplified in a genome-wide screen
to identify host factors required for Zika virus (ZIKV)
infection, which was performed in healthy iPSC-derived
neuronal progenitors (NPs) (Li Y. et al., 2019). ZIKV infection
during early gestation results in reduced differentiation of NPs
and cell death during early cortical development with consequent
fetal brain abnormalities (Mlakar et al., 2016; Tang et al., 2016).
A genome-wide CRISPR KO screen using a pooled library of
187,535 sgRNAs identified host factors that confer resistance
to ZIKV-induced cell death, which were mainly associated
with heparan sulfation, endocytosis, and interferon pathways.
Pharmacological modulation of the identified pathways showed
a protective effect against ZIKV infection in NPs. Among
negative regulators of the interferon pathway, the screen
identified ISG15, in which mutations have been linked to
mycobacterial susceptibility (Bogunovic et al., 2012). The
knockout of ISG15 protected iPSC-derived cerebral organoids
from ZIKV infection in contrast to control cerebral organoids,
consistent with a critical role for the interferon pathway in
ZIKV infection. The comprehensive screen performed on iPSC-
derived NPs identified unknown neuronal-specific pathways
and specific genes involved in ZIKV infection, not previously
highlighted in genome-wide screenings performed on other
cellular models (Savidis et al., 2016). These results underly the
importance of using biologically relevant cellular models for
functional investigations.
On the other side, finding common functional pathways
between a specific set of genes or mutations known to
confer susceptibility to a particular disorder can be useful
for elucidating pathophysiological mechanisms of disease and
to identify possible shared therapeutic targets, limiting the
resources that a genome-wide iPSC-based screen requires. For
example, Deneault and colleagues performed a CRISPR KO of
10 relevant genes that confer susceptibility to ASD (Deneault
et al., 2018) (Supplementary Table 1). Despite gene-dependent
phenotypes, transcriptomic analysis revealed converging effects
with a reduction in synaptic activity in excitatory neurons derived
from CRISPR KOs vs. control line. Although ASD susceptibility
genes belong to different pathways, isogenic iPSC lines revealed
disruption of common signaling networks associated with
neuron projection and synapse assembly and a shared cellular
phenotype, characterized by reduced functional connectivity
of excitatory neurons. Another study focused instead on
familial AD reported a CRISPR-Cas9 based screening of 200
heterozygous disease-causing mutations in amyloid precursor
protein (APP) and presenilin isoforms (PSEN1 and PSEN2)
(Kwart et al., 2019) (Supplementary Table 1). Transcriptomic
and translatomic analyses in cortical neurons derived from the
panel of genome-engineered iPSC lines showed that familial AD
mutations in the two different genes have overlapping effects on
endocytic/endosomal trafficking-associated pathways, previously
associated with late-onset AD. This result suggests that a shared
network of cellular and molecular changes may underlie both
familial and sporadic AD pathogenesis, representing a common
therapeutic target.
CRISPR KO and CRISPR KI screening technology together
with iPSC-based disease modeling can therefore improve
our understanding of pathophysiological mechanisms
and potentially guide therapeutic interventions for
neurological disorders.
CRISPRi/a Screening to Understand
Human Neurological Biology and Disease
States
CRISPRi/a tools allow the modulation of gene expression at the
endogenous transcription level, an advantage compared to RNA
interference (RNAi) and complementary DNA (cDNA) library
screening approaches (Larson et al., 2013; Konermann et al.,
2015).
CRISPRa ability to induce robust expression at specific targets
provides a transformative tool that has been already tested for
the stimulation of neuronal differentiation. Using a mixed pool
of gRNAs directed against either NGN2 or NEUROD1, Chavez
et al. were able to achieve a rapid and robust differentiation
of iPSCs into a neuronal phenotype (Chavez et al., 2015).
Further efforts in the improvement of CRISPRa application for
iPSCs differentiation into neurons and astrocytes focused on
the development of a single all-in-one vector that contained
CRISPR components and modular cassettes for simultaneous
and stable expression of several sgRNAs, achieving a higher
and precise activation of multiple transcription factors than
the one-by-one vectors that relied on co-transfection (Li
et al., 2017). Moreover, the implementation of CRISPRa-based
photoactivatable transcription systems enabled high inducible
activation of endogenous target genes in various human cell
lines and demonstrated to induce NEUROD1 upregulation and
neuronal differentiation from iPSCs (Nihongaki et al., 2017).
CRISPRi-based platforms have been used for the interrogation
of gene function in iPSC-derived glutamatergic neurons (Tian
et al., 2019). Healthy control subject-derived iPSCs have been
engineered for stable expression of both inducible Neurogenin
2 (Ngn2), allowing large-scale production of glutamatergic
neurons, and inducible dCas9-KRAB, to silence target gene
expression. The use of this CRISPRi strategy and downstream
functional readouts highlighted genes essential for neuronal
survival, specific neuronal functions of ubiquitous genes,
and genes involved in maintenance of neuronal morphology.
Black et al., conversely, developed a CRISPRa screening
approach to profile the contribution of putative human
transcription factors to neuronal cell fate specification (Black
et al., 2020). Using single and paired pooled screens, they
identified several proneural factors characterized by a range of
conversion efficiencies, from a clear neurogenic activity to the
requirement of the co-expression with other factors to obtain
a complete differentiation. Interestingly, they identified sets
of transcription factors that improved neuronal differentiation
efficiency, maturation, and subtype specification. These studies
illustrate the utility of CRISPR-based modulation screening
to systematically detect context-specific roles of human genes,
providing a deeper mechanistic understanding of neuronal
physiology and development, and supporting the use of
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unbiased methods to reach a more comprehensive knowledge of
these processes.
CRISPRi/a have also become attractive tools for parallel in-
depth molecular analyses of multiple disease states. The two
technologies have been used to evaluate the feasibility of precise
modulation of the expression of critical neurodegenerative
disease-related genes (Heman-Ackah et al., 2016). Links between
altered expression of pathogenic variants affecting SNCA,
MAPT,HTT, and APP are well established to PD, FTD, AD,
and HD, respectively, which share the common molecular
events of protein misfolding, aggregation, and accumulation,
collectively known as proteinopathies. Overlap of clinical
features is often observed between synucleinopathies and other
neurodegenerative diseases. The first in parallel CRISPRi-
mediated modulation of all aforementioned genes was achieved
in the same cell using a combination of sgRNAs, demonstrating
the feasibility of performing complex manipulations of gene
expression profiles to probe the contributions of specific genes
and combinations thereof to disease phenotypes. This study
demonstrated the possibility of using CRISPRi-based screening
to understand the contribution and possible interplay of different
disease-related genes in a multiplex setting. Moreover, the
study confirmed the possibility to precisely modulate APP
using CRISPRa in iPSC-derived neurons, which could be used
to mimic later-onset degenerative phenotypes, often missing
in iPSC-derived neuronal cultures. This would provide more
realistic modeling of neurodegenerative disorders, avoiding
chemical aging, external stressors, or direct administration/over-
expression of disease-related proteins.
The simultaneous modulation of expression of different
genes through CRISPRi/a gives the possibility to unravel the
contribution of common risk variants to complex genetic
disorders. Schrode et al. (2019) applied CRISPRi/a and
isogenic strategies to manipulate and evaluate endogenous
gene expression of schizophrenia-associated common
variants as discussed in the disease modeling section
(Supplementary Table 1). Therefore, the application of
CRISPRi/a to iPSC models of neurological disorders can
further extend the utility of both systems for the understanding
of pathophysiological mechanisms of disease and may
aid in the development of precision therapies and novel
CRISPR-Cas9-based therapeutic approaches.
RECENT ADVANCES IN GENE EDITING
FOR NEUROLOGICAL iPSC-MODELING
AND FUTURE THERAPIES
CRISPR-based engineering technologies have enabled
researchers to dissect the function of specific genetic elements or
correct disease-causing mutations. In parallel, CRISPR tools are
now being implemented for active control and modulation of
desired messenger RNAs (mRNAs). This allows the interrogation
of transcriptome dynamics and the establishment of causal
links between observed transcriptional changes and cellular
phenotypes. Previously, RNA interference (RNAi) technology
enabled inhibition of desired transcripts using micro RNAs
(miRNAs), but this carried significant off-target effects due to
cross-reaction with targets of limited sequence similarity and
mis-targeting effects linked to endogenous miRNAs (Flynt and
Lai, 2008). Investigation of Cas proteins able to target RNA
led to the development of an engineered RNA-guided and
RNA-targeting enzyme (CasRx) (Konermann et al., 2018), which
showed improved efficiency in knocking down endogenous
mRNA levels compared to RNAi technology, allowing ready
manipulation of alternative splicing in human cells. Moreover,
Konermann and colleagues successfully applied Cas-Rx editing
in a patient-derived cortical neuronal model of FTD to
modulate the balance of tau isoforms. Some forms of FTD with
parkinsonism linked to chromosome 17 (FTDP-17) and other
tauopathies are caused by mutations in the intron following
exon 10 of MAPT (Boeve and Hutton, 2008). These variants
disrupt an intronic splicing site and increase expression of the 4R
tau isoform that contains more microtubule-binding domains
(Kar et al., 2005), inducing pathological changes and driving
the progression of neurodegeneration (Schoch et al., 2016).
CasRx-mediated exon exclusion reduced 4R tau expression to a
level similar to unaffected control neurons, suggesting that this
technology can be exploited for transcriptional modulation in in
vitro models. Interestingly, the small size of CasRx was amenable
to packaging in adeno-associated virus (AAV) for delivery into
post-mitotic neurons, encouraging future clinical applications in
treatment of neurological disorders, and could be paired with an
array encoding multiple guide RNAs for multiplexing. Therefore,
CasRx technology paves the way for transcriptome engineering
and RNA-targeting therapeutic applications.
To date, most functional genomic studies have focused on
protein-coding genes. The increased understanding of the role
of non-coding genome sequences in cellular processes, normal
development, and disease states has promoted the interrogation
of this largely unexplored domain. CRISPR-based editing can
be a useful approach to enable mechanistic studies of specific
non-coding genome functions and complex biology of the
whole genome. Whole-genome screening technology has been
applied in iPSCs and other cell types to interrogate functional
contributions of long non-coding RNA (lncRNA) loci (Liu et al.,
2017). This study considerably increased the number of known
functional lncRNAs essential for cell growth and highlighted the
strong specificity of lncRNA functions in different cell types.
Therefore, cell type-specific complexities in the human non-
coding genome may have important implications for normal
biology and disease states, which should be considered in
both the investigation of pathophysiological mechanisms and
development of therapeutic approaches.
Great efforts are currently directed toward improvements of
CRISPR-based editing, in particular, to increase Cas9 efficiency
that arises from random nucleotide insertions or deletions after
DSBs formation and to reduce off-target effects (Miyaoka et al.,
2016). Increased precision of CRISPR-Cas9 technology holds
great promises for future clinical translation. Base editors (BEs),
for the correction of missense mutations commonly associated
with diseases have recently been developed as an alternative to
the conventional system. BEs enable editing of specific bases
without the induction of DSBs, and they make use of dCas9
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or a nickase version of Cas9 (nCas9) for the generation of
single-filament breaks fused to a deaminase enzyme (Komor
et al., 2016; Gaudelli et al., 2017; Rees and Liu, 2018). More
recently, a modification based on a nCas9-reverse transcriptase
construct, prime editor (PE), has been developed (Anzalone
et al., 2019). PEs have the potential to offer more flexibility,
interconverting any nucleotide or producing small insertions
and deletions, which are commonly reported as disease-causing
variants in human diseases. Both BEs and PEs have been applied
to generate and correct mutations in iPSCs (Sürün et al., 2020).
The study showed that BE is more efficient than nuclease-based
HDR in this cell type, and PE can successfully be applied in
iPSCs for changing more nucleotides simultaneously. Although
improvements in efficiency, specificity, and deliverability for
BEs and PEs are needed for further therapeutic applications
(Anzalone et al., 2020), these new tools offer an opportunity
to further expand genome editing capabilities. However, cell-
state and cell-type determinants are still limiting gene editing.
Therefore, continuous improvement efforts will be crucial to
ensure that these technologies can accomplish their full potential.
THE CHALLENGES AND LIMITATIONS OF
GENOME EDITING IN NEURONAL
DISEASE MODELS
Although the synergy of genome editing and the iPSC system is a
powerful one, there remain inherent challenges for both disease
modeling and CRISPR screens.
These include several fundamental shortcomings of iPSC-
derived cell models as well as issues specific to genome editing.
As discussed, even after long periods in culture, iPSC-derived
neurons only reflect late fetal developmental stages, as shown
by recent advances in electrophysiological and transcriptomic
profiling (Velasco et al., 2019; Logan et al., 2021). While
this is acceptable for early-onset and/or highly penetrant
monogenic disorders with cell-autonomous phenotypes, there
remains concern that later-onset phenotypes or those where
environmental factors are important may not be recapitulated in
vitro. Approaches to mitigate this include chemical cues, progerin
expression, telomere shortening, and direct differentiation where
reprogramming is avoided (Ziff and Patani, 2019). The immanent
variability and heterogeneity of iPSC-derived neurons are due
to a number of factors, including reprogramming-induced
epigenetic changes and genomic instability, genetic background
differences, and differing propensity to differentiation (Soldner
and Jaenisch, 2012). To add to this complexity, myriad
differentiation protocols are in use with significant line to line
and lab to lab variability, raising concerns about reproducibility
(Volpato and Webber, 2020). The use of isogenic controls, robust
quality control using genetic profiling and functional validation
of differentiated cells and single-cell assays such as scRNA
sequencing (scRNA-Seq) will address some of these issues. Lastly,
an undeniable and obvious feature of such in vitro systems is
that they do not represent intact nervous systems or, indeed,
whole brains and therefore cannot model the complex behavioral,
motor and other phenotypes of many neurological disorders.
The major challenge of genome editing in iPSC models is
that of unintended cleavage by site-specific nucleases leading
to off-target effects. These could lead to perturbations of cell
survival or differentiation pathways, which may alter the in
vitro phenotype, as well as modification of the disease-associated
phenotype (Soldner and Jaenisch, 2012). In addition, off-target
effects could lead to genetic alterations of isogenic controls,
confounding phenotypic interpretation (Musunuru, 2013). The
specificity of gene editing in iPSCs has improved due to advances
in Cas9 protein engineering and prime editing (Geng et al., 2020).
Screening for off-target effects with whole-genome sequencing
is not standardized in the field but would lead to a better
understanding and delineation of these effects. The efficiency
of editing of iPSCs was an issue in earlier protocols; this has
improved with the advances in CRISPR/Cas9 discussed above
and with inducible Cas9 (Wang G. et al., 2017), drug selection
for successful clones or the use of electroporation (Geng et al.,
2020).
THE LIMITATIONS OF CRISPR SCREENS
IN iPSC NEURONAL MODELS
GWAS studies reveal a growing list of genetic loci associated
with disease, identified within and outside coding regions, where
they are thought to affect the expression of one or more
coding genes through chromatin accessibility, transcription,
splicing, and mRNA stability regulation (Buniello et al., 2019).
The processes by which these variants contribute to disease
are often unknown or controversial, in particular for complex
disorders influenced by a combination of multiple genetic
and environmental risk factors (Gallagher and Chen-Plotkin,
2018). Mechanistic understanding of disease-linked variants is,
therefore, an important prerequisite for the development of
new therapeutic strategies. iPSCs technology and the generation
of isogenic lines on which allelic effects of a risk variant
can be directly compared definitely increases the detection of
single variant effects. However, resolving the direct biological
consequence(s) of the expanding list of neurological disease-
associated candidate loci needs sensitive and reliable methods
to screen for the functionality of large numbers of variants and
which are scalable to hundreds of SNPs. The parallel analysis
of a wide number of genetic alterations opens up the possibility
to dissect both the contribution of multiple genetic risk factors
to complex disorders and the molecular mechanisms by which
different mutations at same genes or related loci can account
for variable disease phenotypes, with important implications for
therapeutic approaches.
Pooled screens for cell-autonomous phenotypes that can be
monitored through survival or cell sorting for specific cellular
functions followed by next-generation sequencing to determine
sgRNA frequencies should be prioritized given that their
scalability enables a genome-wide approach (Li et al., 2017; Tian
et al., 2019; Black et al., 2020). Pooled screens are more and more
frequently coupled to scRNA-Seq to provide complex and rich
information on cellular processes affected by gene perturbation
(Dixit et al., 2016; Replogle et al., 2020; Schraivogel et al.,
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McTague et al. Genome Editing in iPSC Neural Systems
2020). While high-throughput sequencing of the transcriptome
has rapidly expanded the capability to characterize the effects
of neurological disorders-associated variants, the sheer number
of genetic loci outside coding regions associated with disease
makes it difficult to functionally characterize and validate all
predicted connections. Thus, the successful application of parallel
high-throughput techniques such as CRISPR screens, scRNA-
Seq, scChIP-Seq, and scATAC–Seq, could expand the possibility
for large-scale mapping and investigation of the human genome,
although these readouts are currently expensive to implement.
However, the complex morphology of neurons and glia can
limit the application of pooled screens to phenotypes identified
through optical readout. Moreover, the pooled setup does not
allow the interrogation of non-cell-autonomous phenotypes, in
which the relevant phenotype is not physically coupled to the
cell in which a gene is perturbed, such as excitation of target
cells and interactions between neurons and glia, among others.
Screens for non-cell-autonomous or complex functional or
morphological phenotypes typically require arrayed approaches
that allow setting a multitude of different readouts, such as
high-content imaging (Bolognin et al., 2019; Yamaguchi et al.,
2020) measurements of electrophysiological activity (Daily et al.,
2017), and various biochemical assays (Medda et al., 2016; Zink
et al., 2020). However, arrayed screens necessitate automated
platforms for high-throughput measurement of the phenotypes
of interest, and handling, culturing and analysis of hundreds of
plates, a major limiting factor for most academic laboratories
with limited capacity. Thus, arrayed screens usually entail the
selection of a focused set of genes for perturbation when complex
phenotypes are analyzed (Heman-Ackah et al., 2016; Deneault
et al., 2018; Kwart et al., 2019), restraining the identification of
disease mechanisms and/or new therapeutic targets to already
known disease-associated variants. New advances in automated
platforms for currently labor intensive iPSCs culture and
complex differentiation protocols, both in 2D and 3D (Daily
et al., 2017; Dhingra et al., 2020; Renner et al., 2020; Woodruff
et al., 2020; Boussaad et al., 2021), will help in ensuring
reproducibility of cellular phenotypes and high-throughput
readouts. Technological development will in the future improve
our ability to perform such screens and increase the number
of variants that can be screened still further, expanding our
knowledge about brain physiology and mechanisms that drive
brain disorders with implications for therapeutic interventions.
FUTURE DIRECTIONS FOR GENOME
EDITING IN iPSC NEURONAL SYSTEMS
While the combination of genome engineering and iPSC-derived
neuronal cells has led to many mechanistic discoveries, the driver
for many researchers is the development of novel therapeutic
approaches for these incurable disorders (Figure 4).
Many of these disease models (Supplementary Table 1) have
acted as proof of principle of the rescue of the neuronal
phenotype by gene editing. The knowledge gained from gene
editing in iPSC systems is likely to add to research efforts
to develop regenerative therapies for neurological disorders,
currently most advanced in PD with a trial of transplantation
of HLA-matched iPSC-derived dopaminergic neurons ongoing
(Barker et al., 2017; Stoddard-Bennett and Pera, 2020). If
this is successful, previous gene editing in iPSC PD models
(Supplementary Table 1) could aid the development of edited
patient-derived lines, which may overcome some issues of
immunogenicity and would represent a truly personalized
therapy. However, the most likely translation of insights gained
in iPSC models will be toward in vivo CRISPR gene editing
or promoter modulation. For example, iPSC-derived models of
spinal muscular atrophy demonstrating rescue of the phenotype
by CRISPR-mediated inclusion of exon 7 have paved the way
toward the development of splicing modifiers, such as Risdiplam,
which are now in clinical trials (Poirier et al., 2018). Similarly,
in Duchenne muscular dystrophy, a variety of CRISPR-based
methods have successfully restored dystrophin expression in
iPSC models which was subsequently confirmed in murine
and larger mammal models with current research focusing on
clinical translation (Young et al., 2016; Mollanoori et al., 2020).
iPSC models of Dravet syndrome (Supplementary Table 1),
along with animal models, have contributed to understanding
the cell-intrinsic disease mechanisms of SCN1A dysfunction. A
recent study used CRISPRa to up-regulate SCN1A expression in
interneurons in a Dravet mouse model, rescuing the tendency to
febrile seizures (Colasante et al., 2020).
For translation into the clinic, a number of hurdles still exist
for the majority of CRISPR-based therapeutic strategies. The
first of these is delivery into the central nervous system with
adequate transduction (with efficacy) of target brain regions and
cell types. Viral delivery systems, largely AAV, have been used
in mammal and humans to date, but concerns remain regarding
immunogenicity. While AAVs are preferred to lentiviruses due
to lack of genomic integration, their low packaging capacity is
an issue due to the Cas endonuclease size. Recent advances in
lipid or polymer-based nanoparticles allowing non-viral delivery
hold promise for in vivo therapy (Wilbie et al., 2019). For
example, gold nanoparticles complexed with donor DNA, Cas9
RNP, and the endosomal disruptive polymer PAsp(DET) rescued
dystrophin expression and the muscle phenotype when delivered
in vivo to a mouse model of Duchenne muscular dystrophy
(Lee et al., 2017). The host immune response to the bacterial
Cas protein and the presence of pre-existing immunity due to
microbiome exposure is also a current barrier to translation.
However, the largest hurdle for CRISPR-based therapies is
the risk of off-target events occurring in the host genome.
Better fidelity of gene editing as achieved by recent advances
in CRISPR/Cas9 machinery will reduce this risk. The use of
inducible Cas9 or CRISPR inhibitors such as small molecules
or delivery of Cas9 as a ribonucleoprotein (RNP), rather than
plasmid or viral vectors, can reduce the duration of editing and
thus the risk of unwanted edits (Wilbie et al., 2019).
iPSC models coupled with gene editing are uniquely suited
to help overcome these barriers. One of the major advantages
of iPSC models is that they represent a human platform, which,
combined with gene editing, offers the ability to model many
different mutations in the same model system, as opposed to
laborious generation of transgenic animal models. This means it
is possible to create a personalized system in which to accurately
test new therapies and aim to predict clinical response in vitro
Frontiers in Genome Editing | www.frontiersin.org 13 March 2021 | Volume 3 | Article 630600
McTague et al. Genome Editing in iPSC Neural Systems
FIGURE 4 | Precision medicine. iPSC and gene editing techniques can lead to advances not only in understanding disease mechanisms through in vitro modeling but
also in the development of novel, personalized therapies. Genome editing of patient-derived cells in conjunction with assessment of efficacy and toxicity in in vivo
models allows patient stratification and a tailored approach to treatment.
(Figure 4). For example, the starting iPSC clone can be compared
to gene-edited clones to rigorously assess for off-target effects
with transcriptomics. Recent developments in iPSC modeling,
including 3D systems and the generation of assembloids, such as
fusion of pallial and forebrain organoids (Sloan et al., 2018), are
likely to better recapitulate neuronal interactions and networks
and therefore will represent valuable readouts for assessing the
impact of gene editing. This will also offer the opportunity to test
delivery and targeting into specific cell or tissue types depending
on promoter or nanoparticle delivery. In addition, neurons
can be integrated with non-neuronal cell populations such as
microglia (Marton and Pa¸sca, 2020), which could be integral
in addressing concerns regarding the immunogenicity potential
of CRISPR and AAV systems. The ability to differentiate iPSCs
into cells of any lineage could enable personalized off-target
and toxicity testing in different organ systems, particularly if
systemic administration is envisaged or in multi-system disorders
to assess impact in different tissues. The emerging technology
of personalized “organ on a chip” systems combined with high
throughput transcriptomics is an exciting development that may
have great relevance for testing and translation of precision
therapies (Ronaldson-Bouchard and Vunjak-Novakovic, 2018;
Ramme et al., 2019). Although iPSC-derived systems will not
replace animal models for assessing future genetic treatments’
safety, inter-species differences in sequence homology will impact
targeting strategies and render testing in human-derived models
an important part of the translational process (Wang et al., 2021).
In conclusion, genome engineering has transformed iPSC-
based disease modeling for both Mendelian and more complex
neurological disorders. The increasing accuracy of CRISPR gene
editing, promoter modulation, and epigenome editing coupled
with personalized, patient-derived iPSC model systems now has
the potential for a paradigm shift in our understanding and
treatment of neurological disorders.
AUTHOR CONTRIBUTIONS
AM, GR, AF, SB, and MK conceptual design and writing of the
manuscript. All authors contributed to the article and approved
the submitted version.
FUNDING
AM was funded by the Medical Research Council
(MR/T007087/1) and the Rosetrees Trust (M810). MK, SB,
GR, and AF are funded by the Wellcome Trust (WT098524MA),
NIHR (MK: NIHR-RP-2016-07-019), and the Sir Jules Thorn
Trust (17JTA).
ACKNOWLEDGMENTS
This work is partly funded by the NIHR GOSH BRC. The views
expressed are those of the authors and not necessarily those of the
NHS, the NIHR or the Department of Health.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fgeed.
2021.630600/full#supplementary-material
Frontiers in Genome Editing | www.frontiersin.org 14 March 2021 | Volume 3 | Article 630600
McTague et al. Genome Editing in iPSC Neural Systems
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Conflict of Interest: The authors declare that the research was conducted in the
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