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Abstract

Background:

The main goal of cellular therapy is to effectively engineer the fate of stem/progenitor/terminally differentiated cells into desired lineages to promote therapeutic tissue regenerative effects. Numerous methods such as ectopic transgene overexpression, small molecules, micro RNAs, and CRISPR-Cas9 have been used to engineer cell fate.

Objective:

In this review, we have attempted to highlight various cell fate engineering strategies with a particular emphasis on transdifferentiation that involves CRISPR-Cas9-based approaches in what appear to be the most promising and medically relevant preclinical models.

Methods:

A large number of recent publications involving the application of CRISPR-Cas9-based gene regulation strategies in modulating the identities of different cell types for promoting efficacious tissue regeneration were reviewed.

Results:

From the literature, it appears that the ability to manipulate endogenous gene expression programs has dramatically increased with the help of CRISPR-Cas9-based gene activation/repression/knockout strategies. These approaches have also enabled the generation of cells that closely resemble their true cellular counterparts. Also, in most cases, the efficacy of cell fate engineering through the CRISPR-Cas9-based technology is quite comparable to other methods of cell fate manipulation and in some instances superior.

Conclusion:

The reviewed studies demonstrate novel ways in manipulating cellular identities for regenerative medicine applications using the CRISPR-Cas9-based genome editing tool. Transdifferentiating certain cell types into another using CRISPR-Cas9 seems to have enjoyed more success in comparison to conventional methods. These findings highlight the favorable attributes of the CRISPR-Cas9-based technology in cell-based therapies and their potential use in the near future.

Cell Fate Engineering

Cell fate engineering is the process by which the identity of a stem or a mature cell is manipulated to efficiently differentiate into a particular lineage or acquire a completely new fate. These changes in cell-state can be accomplished by reprograming or transdifferentiation of mature cells or by directed differentiation of stem cells. The ability to reprogram a wide range of mature cells into induced pluripotent stem cells (iPSCs) through the enforced expression of just four transcription factors (TFs), Oct4, Sox-2, Klf4, and c-Myc (OSKM), was a ground breaking and Nobel-Prize-winning discovery in cell fate engineering.1 –3 The controlled differentiation of pluripotent and adult stem cells into desired lineages is an essential cell fate engineering step in generating biomedically relevant cell types in vitro (also in vivo in some cases), which can be transplanted into patients to promote tissue regeneration.4 Transdifferentiation or direct reprograming is an interesting cell fate engineering strategy, which has its roots in a seminal paper published in 1987 when the overexpression of the TF MyoD in mouse fibroblasts gave rise to myocytes.5 In this process, mature cells can be artificially coaxed to undergo (epi)genetic changes into a different cell type without passing through the intervening-induced pluripotent state.6 Reprograming and transdifferentiation have demonstrated that the fate of a cell is malleable and can be manipulated such that the cell can even cross lineage barriers and completely assume the identity of another cell.1,2,7,8 Modulating the expression of lineage specific TFs, which subsequently alters the intrinsic gene regulatory networks, is the most common approach for achieving various cell fate engineering events.8 –10 Ectopic overexpression of TFs has been the de facto procedure for engineering a cell’s fate. The desired factors can be overexpressed through both viral (integrating and non-integrating viruses) and non-viral methods.3,11 Chemical-based methods involving a combination of small molecules that are potential agonists and/or antagonists of key signaling mediators for engineering a cell’s fate have also gained traction in recent years. However, the exact molecular mechanisms behind small molecules-based cell fate engineering events remain largely elusive. A better understanding of these chemical-induced instructive signaling mechanisms responsible for cell fate alterations may enhance the widespread utility of this tool.12 In addition to the above-mentioned techniques, microRNAs (a group of noncoding RNAs) have also been utilized to alter cell identity.13 Among various examples, in vivo transdifferentiation of resident cardiac fibroblasts into functional cardiomyocytes using defined sets of microRNAs could be considered as a rare but remarkable microRNA-mediated cell fate engineering event.14 However, in most cases, microRNA-based cell fate engineering methods appear less robust in altering cellular fate.13

CRISPR-Cas9 for Cell Fate Engineering

The popularly used CRISPR-Cas9 genome editing technology consists of the SpCas9 enzyme (also referred to as Cas9), a DNA endonuclease obtained from Streptococcus pyogenes and is dependent on a single-guide RNA (sgRNA) for genome cleavage.15,16 Many adaptations have been made to the classic CRISPR-Cas9 system through which activation/repression of genes can be accomplished with a high degree of accuracy without deleting the gene of interest using a refined version of dCas9, a mutant form with altered DNA cleaving domains. dCas9 can still bind to the desired DNA sequence directed by the gRNA but cannot create double-stranded breaks. Fusion of dCas9 with various gene regulatory domains enables fine tuning of endogenous gene expression in a precise manner.17,18 Owing to its noncleaving properties, dCas9 fused with various gene regulatory domains has been widely utilized for numerous cell fate engineering events. Some of the regulatory domains that have been used for cell fate engineering are repeats of VP16, VPR, MPH, Sun tag-based system, and Com-KRAB. VP16 is a DNA-binding transcription activator obtained from the herpes simplex virus.19 Multimerization of the VP16 domain can increase the vigor of gene activation.20 VP64, a multimer of VP16, has been reported to improve euchromatin chromatin signatures, namely, H3K27ac and H3K4me3,21 at the target chromatin loci.22 VPR (VP64-p65-Rta) is a trifusion protein designed by conjugating VP64 with the activation domain of p65 (obtained from NF-kB23) and Rta (obtained from gamma Herpesviridae virus family24). It has been reported that merging of p65 and Rta robustly enhances the gene activation potential of VP64.25 MPH gene activation complex is designed by merging p65 and the activation domain of human heat shock factor 1 (HSF1) and is held together by MS2-bacteriphage coat proteins. Guide RNAs harboring MS2 hairpin aptamers are used as a scaffold to which MPH-wt/dCas9 complex can bind and activate the target genes. The combination of dCas9-VP64-MPH-MS2-gRNAs is a strong transcription activator known as synergistic activation mediator (SAM) complex.26 SPH activation domain (Sun tag-p65-HSF1) consists of Sun tag, a protein scaffold that holds p65 and HSF1 activation domain.27 Reports have shown that targeting promoters of desired genes with multiple guides rather than single guide increase the vigor of gene activation.28 –30 Com-KRAB is a gene repressor consisting of RNA binding protein (Com)31 and Kruppel-associated box protein (KRAB), a repression zinc finger32 that disrupts transcription. Guides containing Com binding aptamers are used for recruiting Com-KRAB. Catalytically, active Cas9 conjugated with gene modulating domains but guided by a shorter version of guide RNA known as dead guide RNA or DgRNA is an interesting alternative to dCas9-based strategies. DgRNA is created by removing few nucleotides from the normal guide, which can still steer Cas9 toward the desired genomic location but at the same time prevents it from creating a proper DNA cleavage conformation.33,34 Though clear differences between various CRISPR-Cas9-based gene modulators are yet to be deciphered, it is generally observed that the gene activation potential increases with the addition of activation domains.22,25 Cas9 variants conjugated with gene regulatory domains along with different guides are usually delivered through lentivirus for long-term transduction.35 Plasmid36 and baculoviral vectors37,38 have also shown high efficacy in delivering the Cas9-based gene regulatory components into the target cells. Cas9 variants conjugated with a myriad number of fusion domains can regulate the expression of endogenous genes and alter their epigenetic landscape.18 This remodeling program confers plasticity to the cells and eventually alters their identity. Here, we have discussed how different endogenous gene activation/repression/gene knock out strategies using CRISPR-Cas9 technology has been used to engineer the fate of different cell types. A summary of various cell fate engineering events mediated through CRISPR-Cas systems is provided (Fig. 1; Table 1).

Fig. 1.

A schematic representation of different cell fate engineering events that have been achieved using CRISPR-Cas9-based gene regulation strategies.

Table 1.

A summary of different cell fate engineering strategies performed using CRISPR-Cas system

Source cell	Final fate engineered cell type	Type of CRISPR-Cas system	Transcription factors involved	In vitro setting	In vitro editing followed by transplantation	Direct in vivo cell fate engineering settings	References
CRISPR-Cas9-based gene activation/repression strategies for cell fate engineering
Reprograming
Neuroepithelial stem cells (human)	iPSCs	dCas9-VP192-p65-HSF1 (dCas9-VPH)	OCT4 and EEA	Yes	–	–	Weltner et al.46
Adult fibroblasts (human)	iPSCs	dCas9VPH	OCT4, SOX-2, KLF4, MYC, LIN28A, EEA, and shRNA targeting p53	Yes	–	–	Weltner et al.46
Differentiation
iPSCs and ES cells (human)	Muscle cell progenitors	VP64-dCas9-VP64	PAX7	Yes	Yes	–	Kwon et al.49
iPSCs (human)	Neurons	dCas9-VP64-p65-Rta (VPR)	NGN2/NEUROD1	Yes	–	–	Chavez et al.25
iPSCs (human)	Neurons	padCas9-VP64-MS2-p65-HSF1 (MPH)	NEUROD1	Yes	–	–	Nihongaki et al.50
Mesenchymal stem cells (MSCs) (human)	White adipocytes	dCas9-VP64-MPH	PPARG/CEBPA	Yes	–	–	Furuhata et al.53
MSCs (human)	Beige adipocytes	dCas9-VP64-MPH	PRDM16, PPARG and CEBPB	Yes	–	–	Furuhata et al.53
MSCs (Rat)	Chondrocytes	dCas9-MPH-Com-KRAB	Sox9 and Pparγ	Yes	Yes	–	Truong et al.37
MSCs (Rat)	Osteocytes	dCas9-VP64-MPH	Wnt10b and Foxc2	Yes	Yes	–	Hsu et al.38
MSCs (human)	Sweat gland cells	dCas9-VP64	Ectodysplasin promoter (EDA)	Yes	Yes	–	Sun et al.60
Neural stem cells (mouse)	Oligodendrocytes	dCas9-VP160	Sox10	Yes	Yes	–	Matjusaitis et al.30
Transdifferentiation
Mouse embryonic fibroblasts (MEFs) (mouse)	Oligodendrocytes	dCas9-VP160	Sox10, Olig2 and Nkx6-2	Yes	–	–	Matjusaitis et al.30
Fibroblasts (mouse)	Skeletal muscle cells	VP64-dCas9-BFF-VP64	MyoD1	Yes	–	–	Chakraborty et al.35
MEFs (mouse)	Neurons	VP64-dCas9-VP64	Brn2, Ascl1 and Myt1l	Yes	–	–	Black et al.36
Fibroblasts (human)	Leydig cells	dCas9-VP64-MPH	Nr5a1, GATA4 and DMRT1	Yes	–	–	Huang et al.67
Fibroblasts (human)	Cardiac progenitor cells	dCas9-VP64-MPH	GATA4, HAND2, MEF2C, and TBX5	Yes	–	–	Wang et al.71
Astrocytes (mouse)	Neurons	dCas9-SPH	Ascl1, neurod1 and neurog2	–	–	Yes	Zhou et al.27
Hepatocytes (mouse)	Pancreatic beta cells	Cas9-MPH	Pdx-1	–	–	Yes	Liao et al.75
CRISPR-Cas9-based gene knock out strategies for cell fate engineering
Transdifferentiation
Myoblasts (mouse)	Brown adipocytes	Cas9	MyoD	Yes	Yes	–	Wang et al.83
Rod cells (mouse)	Cone cells	Cas9	Nrl	–	–	Yes	Yu et al.86 and Zhu et al.87
Use of different Cas proteins for cell fate engineering
Differentiation
Umbilical cord-derived MSCs (human)	Osteocytes	dCpf1-VPR	BMP4	Yes	–	–	Choi et al.90
Transdifferentiation
Astrocytes (mouse)	Dopaminergic neurons	CasRx	Ptbp1	–	–	Yes	Zhou et al.94
Muller glial cells (mouse)	Retinal ganglion cells	CasRx	Ptbp1.	–	–	Yes	Zhou et al.94

Computational Approaches in CRISPR-Cas9-based Cell Fate Engineering

TFs in close communication with epigenetic modifiers are chiefly responsible for governing the fate of a cell throughout its lifespan.39 With the advent of iPSCs through OSKM TF-mediated reprograming, various combinations of TFs have been utilized to transform a wide range of cell type fates. Given their vast numbers, narrowing down the specific TFs in the right combination is essential to induce morphological, physiological, and (epi)genetic changes in a cell to achieve the desired cellular plasticity. TFs responsible for fate specification have been predominantly deciphered through extensive labor-intensive experiments. In vivo transdifferentiation, a more preferable tissue regenerative approach, is still in its infancy owing to the lack of sheer knowledge in determining the optimum set of TFs expressed at the right levels.40 To facilitate the identification of optimal sets of TFs for cell-cell conversion, various high throughput bioinformatics approaches have been developed by several groups. Morris et al.41 devised a systems biology tool called CellNet, which map essential gene–gene interactions from a vast repertoire of gene expression data to identify the putative players responsible for maintaining a particular cell’s identity. TFs predicted by CellNet were used to transdifferentiate B cells into macrophages and fibroblasts into hepatocytes.41 Rackham et al.42 employed a state-of-the-art bioinformatics approach known as Mogrify. It gathers previously available transcriptome data from the functional annotation of the mammalian genome-FANTOM5 and predicts the optimal combination of TFs for achieving various transdifferentiation events. Two different de novo conversions utilizing the factors identified through Mogrify were also experimentally verified. This involved the transdifferentiation of human fibroblasts into keratinocyte-like cells and keratinocytes into vascular endothelial cells. Both of the transdifferentiated cells exhibited appropriate physical and molecular features demonstrating the faithfulness of the algorithm. Mogrify has identified TFs for innumerable plausible transdifferentiation strategies that can be retrieved from online resources.42 Furthermore, dCas9-based screening strategies have been utilized to determine the optimum sets of TFs responsible for neuronal fate engineering by utilizing mouse embryonic stem cells (ESCs)/iPSCs. The factors identified enabled the construction of a gene interaction map involved in promoting neuronal fate. The gene activation screens also aided in uncovering numerous novel surrogate factors involved in enhancing the differentiation of ESCs/iPSCs into neurons.43,44 CRISPR-Cas9 screening strategies utilizing the predicted factors from various bioinformatics tools will enable researchers to determine the optimal set of cell fate engineering factors that may be useful to achieve direct in vivo cell fate alterations for therapeutic purposes in the future (Fig. 2).

Fig. 2.

A futuristic view demonstrating the application of computational tools and CRISPR-Cas9-based screening strategies to determine the most optimum sets of factors that could be used for direct in vivo conversion of supportive stromal cells into cells of therapeutic importance.

CRISPR-Cas9-based Gene Activation/Repression Strategies for Cell Fate Engineering

Reprograming neuroepithelial stem cells and fibroblasts into iPSCs

iPSCs have been previously created using transgene overexpression or through induction with small molecules.3,45 Weltner et al.46 demonstrated that endogenous activation of OCT4 and EEA motif (human embryo genome activation) in human neuroepithelial stem cells (derived from iPSCs) using dCas9-VP192-p65-HSF1 (dCas9VPH) activation complex robustly reprogramed them into hallmark iPSCs. Furthermore, the same group also reprogramed adult human skin fibroblasts into iPSCs by transfecting them with dCas9VPH, an shRNA targeting TP53, and guides targeting activation of OCT4, SOX-2, KLF4, MYC, LIN28A, and EEA. These iPSCs were functionally and genetically identical to the ESCs and iPSCs generated through non-CRISPR methods.

Lineage directed differentiation of ESCs/iPSCs into myogenic progenitor cells

Kwon et al.49 differentiated human iPSCs/ESCs into myogenic progenitor cells (MPCs) by transducing them with VP64-dCas9-VP64, guides targeting PAX7 promoter, and stimulating the culture with the GSK-3 inhibitor-CHIR99021. Differentiation dynamics was compared with MPCs generated through PAX-7 overexpression in ESCs/iPSCs. It was observed that the MPCs generated through dCas9-mediated gene activation had stable expression of PAX7 (muscle stem cell marker47) conserved through multiple generations in comparison with the ectopic PAX7 overexpression group. Also, only the MPCs generated through endogenous PAX7 activation had enhanced presence of active gene expression markers, H3K27ac and H3K4me3,21 in the PAX7 promoter. Furthermore, these MPCs repopulated the muscle stem cell compartment of the barium chloride-mediated muscle injury48 mouse model much more vigorously than the myoblasts obtained through the PAX7 cDNA overexpression approach. RNA sequencing analysis disclosed that the MPCs generated through dCas9-mediated activation exhibited gene expression much more specific for the formation and differentiation of muscle stem cells in comparison with MPCs from PAX7 overexpression.49 This outcome demonstrates the superior attributes of CRISPR-Cas9 in differentiating ESCs/iPSCs into MPCs through endogenous gene activation, and thus raising the possibility of generating clinical grade MPCs from patient-specific iPSCs that may closely resemble their true counterparts for muscle cell-based therapies.

Lineage directed differentiation of iPSCs into neurons

Chavez et al. differentiated human iPSCs into neurons by transducing them with dCas9-VPR (VP64-p65-Rta) and guides targeting either of the two neuronal TFs NGN2 or NEUROD1.25 Similarly, a de novo photoactivatable dCas9 or padCas9 developed by Nihongaki et al. was employed for differentiating human iPSCs into neurons. Electroporation of padCas9, VP64-MPH, and guides targeting NEUROD1 into iPSCs followed by continuous exposure to blue light for 4 days induced the formation of neurons expressing β-III tubulin. The iPSCs remained undifferentiated in the absence of blue light, proving that the system is tightly regulated by photo stimulus.50 This area of photo-stimuli-controlled cell fate engineering might be a useful tool for modulating the expression of cell identity altering factors, wherein other methods of systemic control may not be ideal or possible.

Differentiation of mesenchymal stem cells into different cell types

Mesenchymal stem cells (MSCs) are multipotent in nature and can give rise to adipocytes, chondrocytes, osteocytes, and myocytes, among others. Efficient differentiation of MSCs into desired cell types is a pivotal factor for effective and safe cellular therapies.51,52

Furuhata et al.53 differentiated human MSCs into white adipocytes by transducing them with dCas9-VP64-MPH (SAM complex) and guides activating PPARG/CEBPA. Transducing MSCs with SAM complex and guides for PRDM16, PPARG, and CEBPB differentiated them into beige adipocytes.53 This has given insights that endogenous gene activation through CRISPR-Cas9 technology enables the generation of two closely but distinct cell types from a common progenitor just by changing the combination/specificity of the guides. This might be beneficial for strategic therapeutic purposes, wherein one type of adipocyte is exclusively preferred over the other.

Truong et al. utilized dCas9 gene modulation technology for heightening the differentiation of MSCs into chondrocytes. dCas9 coupled with MPH and Com-KRAB was used for upregulating chondrogenesis promoter (Sox-954) and suppressing adipogenic promoter (Pparγ55), respectively. dCas9-MPH-Com-KRAB complex along with the targeted guides was delivered into MSCs through a baculovirus vector expression system. MSCs efficiently differentiated into chondrocytes with elevated levels of various chondrocyte markers and effectively formed cartilage in 3D culture. Furthermore, bone regeneration occurred more effectively in calvarial (skull bone) injury rat models transplanted with gene-edited MSCs compared to the models that received mock transduced MSCs.37 Although calvaria bones are normally formed through intramembranous ossification,56 this particular regeneration event was based on the concept that damaged skull bones could be repaired through endochondral ossification. It is a process that utilizes a chondrocyte templates for promoting bone formation and has enjoyed previous success in improving skull bone defects.57

Similarly, Hsu et al.38 utilized the dCas9 gene activation system for effective differentiation of MSCs into osteocytes. It was orchestrated by the activation of Wnt10b58 and Foxc259 (activators of Wnt signaling for promoting osteogenesis) in rat BM-MSCs mediated by dCas9-VP64-MPH complex. Transplanting gene-edited MSCs into the injured calvaria of rats augmented the regeneration of the bones and reduced the size of the defect area compared to the rats that received mock-transduced BM-MSCs.38 The two studies mentioned earlier employed the baculovirus system containing two vectors for delivering the CRISPR-Cas9 gene regulatory complex. The main vector consists of the entire gene regulatory complex flanked with loxP sites on both the ends, whereas the other vector contains the Cre enzyme. This strategy converts the main loxP containing vector into an episome through Cre recombinase, thus sustaining the activity of gene regulatory components inside the cell for an extended period of time and also escaping from the host cell’s immune reaction against the vector particles.37,38

Human BM-MSCs were efficaciously differentiated into sweat gland (SG) cells using the CRISPR-Cas9 activation system. Sun et al.60 illustrated that transduction of BM-MSCs with dCas9-VP64 and guides activating ectodysplasin promoter (EDA) induced the formation of SG-like cells. Furthermore, transplantation of these gene-edited BM-MSCs into paws of mice with destroyed SGs hastened the reconstruction of the epithelial layers and increased the number of SGs in comparison with the group that received only mock-transduced MSCs. Clinical safety evaluation revealed the absence of tumor formation after the transplantation of the gene-edited BM-MSCs, suggesting their safety for potential clinical use.60

Ex vivo editing of MSCs using CRISPR-Cas9 technology followed by transplantation into animals demonstrated accelerated wound healing in comparison with the group that did not receive the gene editing complex. Though more comprehensive analysis is required before moving onto clinical trials, editing MSCs with CRISPR-Cas9 seems to have propitious healing effects.

Differentiation of neural stem cells into oligodendrocytes

The dCas9 activation system was used by Matjusaitis et al.30 to improve the differentiation of neural stem cells (NSCs) into oligodendrocytes (OL). Co-transfection of adult mouse NSCs with PB transposase, a plasmid consisting of dCas9-VP160, guides targeting Sox-10, and piggybac (PB) transposase recombination sites enforced differentiation into OLs.30 The PB transposase system was used for effective genome integration. These cells expressed twice the level of various OL markers compared to the differentiating NSCs that did not receive the dCas9 gene regulatory complex. Furthermore, these gene-activated NSCs contributed to the formation of myelin sheaths when transplanted into shiverer mice,61 which lack the ability to form myelin due to a mutation in myelin basic protein (MBP).

Transdifferentiation of fibroblasts into oligodendrocytes

Matjusaitis et al.30 transfected primary mouse embryonic fibroblasts (MEFs) with the same CRISPR complex as described before30 along with gRNAs targeting Sox-10, Olig2, and Nkx6-2, which transdifferentiated them into near OLs. However, engraftment of these OLs into the brain of the shiverer mouse did not generate myelin sheaths though they gave rise to MBP-producing cells. It should be noted that OLs generated from fibroblasts through transgene overexpression were functional and generated myelin sheaths in vivo.62 This indicates that the activation of additional factors might be required in order to achieve robust transdifferentiation of fibroblasts into OLs through CRISPR-Cas9 system. Nonetheless, these results show how endogenous gene activation strategies could be used in two different systems for generating OLs albeit with low efficiency from MEFs.

Transdifferentiation of fibroblasts into myocytes

Fibroblasts were transdifferentiated into skeletal muscle cells by Chakraborty et al. (2014) using a doxycycline (Dox) inducible version of dCas9 containing VP64 at its two terminals. Blue fluorescent protein (BFP) was coalesced with dCas9 activation complex for tracking. VP64-dCas9-BFP-VP64 activation complex was used in two different fibroblast systems to alter them into myocytes. Lentiviral transduction of C3H10T1/2 (a multipotent cell line with the characteristics of fibroblasts63) with the gene activation dCas9 complex and guides targeting MyoD1 followed by dox induction transdifferentiated them into muscle cells. These cells possessed multinuclear myotube formation with genetic characteristic features of sarcomere. Similar results were obtained when the same methodology was repeated in freshly obtained MEFs. Moreover, myocytes generated through dCas9-mediated activation had elevated levels of MyoD1 in comparison with the muscle cells generated through transgene overexpression giving credence to the CRISPR-Cas9 system.35

Transdifferentiation of fibroblasts into neurons

Ectopic overexpression of Brn2, Ascl1, and Myt1l (BAM factors) in fibroblasts has been reported to transdifferentiate them into neurons.64 Black et al.36 transdifferentiated MEFs expressing VP64-dCas9-VP64 into neurons by transfecting them with guides for activating endogenous BAM factors. Transdifferentiation dynamics was compared between dCas9-mediated BAM factors activation and plasmid-based BAM factors overexpression in MEFs. It was observed that the levels of BAM factors increased swiftly and remained at high levels throughout the course of the experiment only in the group that received dCas9 activation complex in comparison with the plasmid-based BAM overexpression group. Moreover, active gene expression marks H3K4me3 and H3K27ac21 were observed only in the Brn2 and Ascl1 locus of the neurons generated through endogenous gene activation and not in the neurons generated through plasmid-based BAM overexpression. It should be noted that efficiency of neuronal generation was higher when BAM factors were overexpressed through lentiviral transduction. Nevertheless, this study suggests that CRISPR-Cas9-based short-term induction of endogenous genes may be an efficacious approach for transdifferentiating certain cells types.36

Transdifferentiation of fibroblasts into leydig cells

Previously, fibroblasts have been transdifferentiated into functional leydig cells (LCs) using cDNA overexpression or through a combination of small molecules.65,66 Huang et al.67 transdifferentiated fibroblasts into LCs through dCas9-mediated endogenous gene activation. Infection of human fibroblasts with steroid-specific receptor 3β hydroxysteroid dehydrogenase, dCas9-VP64-MPH complex, and guides targeting LC specific genes, Nr5a1, GATA4, and DMRT1, through lentivirus mediated the transdifferentiation.67 These cells produced testosterone when induced with the human chorionic gonadotrophin hormone68 and were positive for various LC markers. Epigenetic analysis revealed the increased presence of euchromatin marks, H3K4me3 and H3K27ac,21 in the LC specific genes. Even though standardization of the protocol is required for effective testosterone production, this result demonstrates how endogenous gene activation can orchestrate complex transdifferentiation.

Transdifferentiation of fibroblasts into cardiac progenitor cells

Cardiac progenitor cells (CPCs) have been previously generated from fibroblasts through transgene overexpression or with the help of small chemical molecules.69,70 Wang et al.71 activated endogenous cardiac genes using CRISPR-Cas9 system in human fibroblasts to transdifferentiate them into CPCs. Transduction of fibroblasts with dCas9-VP64-MPH and guides targeting the cardiac factors GATA4, HAND2, MEF2C, and TBX5 (GHMT) orchestrated the transdifferentiation event.71 The induced CPCs exhibited elevated mesodermal markers with a stark downregulation in the expression of fibroblast markers during the course of transdifferentiation. Furthermore, the CPCs were functional and in vitro settings gave rise to cardiomyocytes, smooth muscle cells, and endothelial cells of cardiac lineage asserting their differentiation capabilities. Epigenetic analysis revealed the enhancement of H3K4me321 in the cardiac specific genes: TNNT2 and ACTC1.72 Although the in vivo potential of these CPCs is yet to be deciphered, this study demonstrates the capability of CRISPR-Cas9 in engineering the fate of easily sourced cell such as fibroblasts into CPCs.

Transdifferentiation of astrocytes into neurons

Zhou et al.27 developed a transgenic mouse line expressing dCas9 and SPH gene activation complex (Sun Tag-p65-HSF1) only in astrocytes. Adeno-associated viral (AAV) delivery of gRNAs targeting the neural factors Ascl1, Neurod1, and Neurog2 into one part of the mid brain of these mice robustly transdifferentiated astrocytes into neurons compared to the other part of the mid brain that received only control constructs. Examination of brain slices from the gene-edited group revealed that the newly formed neurons were able to form synapses, respond, and propagate the electrical impulse at the neuronal junctions.27 Though in vivo transdifferentiation of astrocytes into neurons has been previously achieved through transgene overexpression method,73 this example demonstrates how endogenous gene activation through dCas9 gene regulatory complex can nearly accomplish the same feat.

Transdifferentiation of hepatocytes into pancreatic beta cells

Liao et al.75 utilized Cas9-MPH complex conjugated with DgRNA for transdifferentiating hepatocytes into pancreatic β-cells completely in vivo. AAV delivery of MPH-MS2-DgRNA targeting Pdx1 (a key gene determining the fate of pancreatic progenitors74) into the liver of type 1 diabetic mice expressing Cas9 transdifferentiated the hepatocytes into functional insulin producing pancreatic β-cells expressing pertinent markers. Most importantly, this in vivo transdifferentiation event reduced the levels of glucose in the blood with the concurrent increase in the blood insulin levels compared to mock construct-injected mice.75 Though there are several roadblocks that have to be overcome before moving onto clinical trials, this study suggests the potential of CRISPR-Cas9-based in vivo transdifferentiation therapy for the most notorious of metabolic disorders diabetes.

CRISPR-Cas9-based Gene Knock Out Strategies for Cell Fate Engineering

Transdifferentiation of myoblasts into brown adipocytes

Brown adipose tissue (BAT), beige adipocytes, and white adipose tissue (WAT) are the three major types of mammalian fat tissue. BATS and beige adipocytes are closely related and possess similar functions, whereas BATs and WATs are warring cousins which work in opposition to each other. WATs are the storage reservoirs of surplus fat, while BATs break down the excess fats and maintain the thermostability of the body.76,77 Studies performed in rodents have shown that BATS confer resistance against metabolic disorders such as obesity and diabetes by exhausting the over abundant lipid content of the body.78 Harnessing BAT’s thermogenesis properties is considered to have tremendous therapeutic potential in treating various metabolic disorders.77 Muscle cells possess many characteristics in common with BATs and are an excellent source for generating them. Previously, it has been shown that muscle cells could be transdifferentiated into BATs by inducing them with specific TFs or small chemical molecules.79,80 Wang et al.83 illustrated that the loss of MyoD (an important factor involved in myocyte development81) in C2C12 cells (a mouse skeletal myoblast cell line⁸²) through Cas9-mediated cleavage transdifferentiates them into BATs.83 The newly formed BATs secreted oil droplets and had elevated the expression of adipogenic markers. Furthermore, these BATs exhibited an intense upregulation of PI3K-Akt pathway, a critical pathway involved in adipogenesis.84 Implantation of the MyoD knock out C2C12 cells into the trauma-induced dystrophin negative muscles of mice preferentially gave rise to fat forming adipocytes compared to the mice injected with unedited C2C12 cells. This result further substantiated that the loss of MyoD transdifferentiates myoblasts into adipogenic lineage even after transplantation. Despite the fact that additional experimentations are required to confirm various safety parameters, this result gives an insight on futuristic Cas9-based transdifferentiation therapy for treating various metabolic disorders.

Transdifferentiation of rod photoreceptors into cone cells

Retinitis pigmentosa (RP) is a genetic disorder affecting both the rod and cone cells of the retina, leading to their degeneration which consecutively leads to various degrees of vision loss. Degradation of cone cells resulting from the loss of rod photoreceptors is the main reason for total blindness in RP individuals.85 It is, thus, very crucial to prevent cone cell loss, which can be prevented only by abating the root cause, that is, rod cell loss. As an innovative strategy, two independent groups Yu et al.86 and Zhu et al.87 published similar results on a gene inhibition–gene therapy-based approach, in which the mutation prone rod cells become non-responders to RP-related mutations and become resistant to the disease. This was achieved by the transdifferentiation of rod cells into cones which eventually prevents damage to cones. Injection of an AAV vector carrying Cas9 with guides for disrupting Nrl (regulator of rod specification88) into the retina of RP mice models transdifferentiated some rod receptors into cone marker-expressing cells with the dramatic decrease in the rod-specific genes. Functional analysis revealed an increase in the electrophysiological properties of the cone cells. This transdifferentiation event decelerated retinal degradation and improved the vision of the RP mice models in comparison with the controls that received either Cas9- or Nrl-targeting guides alone. This study suggests the capability of CRISPR-Cas9-mediated gene editing therapy for abating photoreceptor degradation.

Use of Different Cas Proteins for Cell Fate Engineering

Differentiation of MSCs into osteocytes

A relatively newer type of nuclease Cpf1 also known as Cas12a89 sourced from Acidaminococcus sp. BV3L6 was used by Choi et al.90 for differentiating MSCs. Delivery of nuclease dead version of Cpf1 (dCpf1), VPR (VP64-p65-Rta), and guides targeting endogenous bone morphogenic protein 4 (BMP4) into human umbilical cord-derived MSCs differentiated them into osteocytes.90 The differentiated osteocytes stained positive for alizarin red, an indicator of osteocytes along with the presence of various osteocyte markers. It was also observed that the Cpf1-VPR system has higher gene activation potential in upregulating BMP4 when compared with the dCas9-VPR system.

Transdifferentiation of glial cells into different types of neurons

Loss of dopamine-producing neurons is a hallmark feature of Parkinson’s disease (PD), causing various cognitive and motor dysfunctions.91 The current treatment strategies employing l-dopa alleviate some of the symptoms but do not completely reverse the impact of the disease.92 In a quest to develop an efficient therapeutic approach, Zhou et al.94 utilized CasRx93 (called as Cas13d belonging to Cas13 family), an RNA-guided RNA cleaving protein for depleting a particular gene’s mRNA to ameliorate the pathological features of neurological disorders like PD and vision loss. Knock down of polypyrimidine tract binding protein 1 (Ptbp1) in striatum astrocytes from a PD mouse model through CasRx-mediated RNA targeting transdifferentiated them into functional dopamine-producing neurons in the midbrain substantia nigra region. These neurons stained positive for various dopaminergic neuron markers demonstrating their stalwart attributes. The cells also displayed robust proficiency in electrical properties by responding well to depolarizing and hyperpolarizing currents. Most importantly, these functional dopamine-producing neurons were able to attenuate the motor dysfunction and the behavioral aspects of PD mice models. Similarly, knock down of Ptbp1 by CasRx in muller glia of retinal injury mice models transdifferentiated them into retinal ganglion cells (RGCs) possessing new axon projections. The RGCs responded to light demonstrating the significant improvement in the vision of the mice.94 Notably, the improvement of symptoms in both the experiments was seen only in the animals that received the CasRx-Ptbp1 constructs and not in the control CasRx alone injected group. In a different scenario, CasRx-mediated knock down of Vegf in age-related macular degeneration mouse models dramatically stopped neovascularization in the eyes without affecting the retinal architecture.95 These studies suggest the very real prospects of CasRx for fine-tuning therapeutic gene downregulation strategies and may have less off-target effects.93,96,97

Cell Fate Engineering, CRISPR-Cas9 Technology, and Future Perspectives

A lot of emphasis has been given in recent years to study various aspects of cell fate engineering in order to create efficient regenerative therapies. Particularly, transdifferentiation of resident cells present nearby damaged tissue into cells of therapeutic importance is viewed as a very important approach to tissue regeneration that needs further in vivo validation. In vivo transdifferentiation reduces the laborious task of transplanting cells into more complex areas of the body.40 One concern that is associated with this strategy is generating sufficient levels of physiologically relevant cells, which are free from epigenetic memory of original cell and that also maintain their cellular identity in a sustained manner.98 Additional investigations are required to know if in vivo transdifferentiation of resident cells may disrupt the function of the tissue in which it is present.99 Transdifferentiation of highly proliferating cells would be an ideal strategy to generate cells of therapeutic importance and also preserve supporting stromal cells. One other option is to specifically target the cells in scar tissue formed in various disorders such as myocardial infarction14, stroke,73 or skin wounds100 and transdifferentiate them into relevant stem cell types. It could be seen from the various examples that not only activation of certain genes enables transdifferentiation but also knock down of even a single gene can orchestrate direct transition into a different cell state. These genes might be the guardians of cellular identity of the particular cell type and their loss may allow the cell to switch fates.83,86,87,94 Understanding the intrinsic gene regulatory networks controlled by these genes may also enable novel therapeutic transdifferentiation approaches. The advent of single cell transcriptomic data has greatly facilitated our understanding of transdifferentiation dynamics that fibroblasts undergo when transforming into neurons101 and cardiomyocytes102 –104 at the single cell level. These studies demonstrate the huge potential of omics data in helping to decipher the various different transdifferentiation events at the single cell level with high fidelity. This new data will undoubtedly advance the quest to develop more effective cellular therapies.

From the above discussed examples, it is clear that the efficiency of various cell fate engineering strategies through intrinsic modulation of endogenous gene networks is quite comparable to the conventional TF overexpression approaches. Indeed, transdifferentiating fibroblasts into myocytes and neurons through CRISPR-Cas9-based approaches has enjoyed a greater degree of success in comparison with conventional ectopic TF overexpression methodologies.35,36 Furthermore, editing MSCs with CRISPR-Cas9 has also shown to improve its tissue regeneration potential.37,38,60 Emerging reports have also elucidated various successful in vivo transdifferentiation events orchestrated with the help of CRISPR-Cas9.27,75,86,87 Simultaneous activation and repression of key signaling mediators could be achieved with the help of CRISPR-Cas9, suggesting that complex cell fate engineering strategies might be performed with ease.37 However, several factors such as potential immunogenic responses associated with endogenous Cas9 expression,105 efficacious delivery of the gene regulatory complex106 and potential off target effects107 must be carefully evaluated in the context of cell or endogenous gene-based therapies.

Conclusion

The dawn of the CRISPR era has brought a new outlook to biomedical research with some hopes from the CRISPR-Cas9-based cancer CAR-T-cell-based immunotherapy trials in humans. The first trials started in 2019, wherein T cells were genetically modified using CRISPR-Cas9 in order to attack cancer cells with heightened vigor. Furthermore, in early 2020, the first clinical trial on CRISPR-Cas9-based in vivo gene therapy was done on a patient suffering from Leber congenital amaurosis (an eye disorder), which aimed to therapeutically edit a point mutation responsible for driving the pathology of the disease. It is a promising system for achieving tailor-made therapies especially through transdifferentiation. Temporal and spatial control of Cas9 allows users to optimize cell fate engineering with greater efficiency. Even though numerous safety parameters concerning various in vivo cell fate engineering strategies and CRISPR-Cas9 technology have to be examined thoroughly on a large scale before moving onto the next phase, the aforementioned studies serve as a beacon in the tempest field of cell-based therapies.

Authorship confirmation statement: VK and JH planned, wrote, and edited the manuscript. All authors have reviewed and approved the final manuscript. The manuscript has been submitted solely to this journal and is not published, in press, or submitted elsewhere.

Author's disclosure statement: The authors declare no competing financial interests exist.

Funding statement: JH was supported by Canadian Institutes of Health Research (CIHR) research grant and CancerCare Manitoba Foundation grant. VK was supported by a studentship from Research Manitoba.

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CRISPR-Cas9—The Potential “Holy Grail” for Generating Biomedically Relevant Cells through Cell Fate Engineering

Abstract

Abstract

Background:

Objective:

Methods:

Results:

Conclusion:

Cell Fate Engineering

CRISPR-Cas9 for Cell Fate Engineering

Computational Approaches in CRISPR-Cas9-based Cell Fate Engineering

CRISPR-Cas9-based Gene Activation/Repression Strategies for Cell Fate Engineering

Reprograming neuroepithelial stem cells and fibroblasts into iPSCs

Lineage directed differentiation of ESCs/iPSCs into myogenic progenitor cells

Lineage directed differentiation of iPSCs into neurons

Differentiation of mesenchymal stem cells into different cell types

Differentiation of neural stem cells into oligodendrocytes

Transdifferentiation of fibroblasts into oligodendrocytes

Transdifferentiation of fibroblasts into myocytes

Transdifferentiation of fibroblasts into neurons

Transdifferentiation of fibroblasts into leydig cells

Transdifferentiation of fibroblasts into cardiac progenitor cells

Transdifferentiation of astrocytes into neurons

Transdifferentiation of hepatocytes into pancreatic beta cells

CRISPR-Cas9-based Gene Knock Out Strategies for Cell Fate Engineering

Transdifferentiation of myoblasts into brown adipocytes

Transdifferentiation of rod photoreceptors into cone cells

Use of Different Cas Proteins for Cell Fate Engineering

Differentiation of MSCs into osteocytes

Transdifferentiation of glial cells into different types of neurons

Cell Fate Engineering, CRISPR-Cas9 Technology, and Future Perspectives

Conclusion

References