Introduction {#S1}
============

Stem cells are defined by their long-term self-renewal and their ability to differentiate into specialized progeny ([@B131]). Pluripotent stem cells (PSCs) can adopt any cellular fate from the three germ layers (ecto-, meso-, and endoderm) ([@B141]). Based on their tissue of origin, stem cells can be classified as embryonic (ESC), which are collected from the inner cell mass of the blastocysts ([@B85]), or induced pluripotent (iPSC), which are somatic cells reprogrammed toward a more primitive state ([@B114]). Multipotent stem cells, such as neural stem cells (NSC) and mesenchymal stem cells (MSC), have more limited differentiation capacity but can still generate multiple cell types ([@B174]; [@B39]). Adult stem cells are found in various tissues in the body and serve to replace any tissue lost due to damage or injury ([@B169]).

The advent of genome engineering tools like Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) and most recently the Clustered Regularly Interspaced Short Palindromic Repeats Palindromic Repeats (CRISPR) Cas systems have vastly expanded our ability to modify gene expression ([@B108]; [@B166]). In particular, the CRISPR Cas system has revolutionized our ability to perform gene knockout and gene regulation with a high degree of specificity with Cas9 (CRISPR associated protein 9) and dCas9 (deactivated Cas9), respectively ([@B40]). The relative simplicity of this technique has facilitated the study of stem cells in a context dependent manner, particularly using more physiologically relevant 3D cell culture platforms.

Although, early embryonic development has been broadly studied using conventional two-dimensional, cell monolayer platforms ([@B22], [@B23]; [@B98]) these systems do not fully recapitulate *in vivo* behavior ([@B90]; [@B44]; [@B116]). Conversely, three-dimensional (3D) systems, such as spheroids and organoids, demonstrate a high degree of maturation and functionality. These structures are often generated by the formation of stem cell aggregates known as embryoid bodies ([@B135]). This is due to more *in vivo*-like signaling and biophysical gradients, as well as cell--cell contacts, that can impact the many transcriptional networks ([@B154]; [@B16]) and metabolomic pathways inside the cell ([@B51]; [@B29]).

Herein, we focus on the application of CRISPR Cas9 tools to knockout/knock-in genes in pluripotent and adult stem cells for disease modeling. A broad overview is provided on the generation of 3D models and a mechanistic understanding of the CRISPR Cas9 technology, and this is followed by the use of gene knockouts to interrogate human diseases. Key to the latter is how CRISPR Cas9 has been used to develop physiologically relevant disease models that affect multiple organ systems. Finally, existing gaps are addressed leading to opportunities for future research.

Engineering Strategies for 3D Organoid Models for Stem Cells {#S2}
============================================================

The current definition of an organoid is a self-organized, 3D multicellular structure that possesses functional attributes to its *in vivo* counterpart ([@B146]). While organoids have been examined for decades in tumor-derived explants ([@B148]), more recently, organoids have been developed to include self-organized structures derived from pluripotent and adult stem cells. This self-organization is intrinsically dependent on biochemical factors like morphogens, small molecules and growth factors that are delivered in a spatiotemporal fashion as well as on biophysical stimuli provided by cell--cell and cell-extracellular matrix (ECM) interactions (extensively reviewed in [@B17]; [@B145]).

Several protocols have been established to promote cellular assembly of 3D structures to recapitulate organ level functions *in vitro* with both scaffold-based and scaffold free approaches as shown in [Figure 1](#F1){ref-type="fig"}. In scaffold-based approaches, the microenvironment of naive tissue is provided by matrices that replicate specific *in vivo* physical and biochemical stimuli. Early approaches relied on naturally derived matrices from decellularized tissue ([@B45]). For example, [@B136] used laminin-rich Matrigel as an encapsulating matrix to support epithelial growth of mouse intestinal crypts. An alternative approach involved an air-liquid interface that provides better oxygenation to 3D intestinal cell cultures ([@B120]). In this study, a collagen matrix was used to encapsulate primary intestinal cells in the presence of myofibroblast, which provided essential cues to recapitulate an intestinal stem cell niche allowing cell growth and differentiation with the additional external delivery of WNT and Notch signaling molecules. In the context of hPSC, [@B92] and [@B91] have developed a widely used approach, in which cerebral organoids were prepared for modeling microcephaly via knockdown RNA interference (iRNA) on hiPSC lines with disease-associated Cyclin Dependent Kinase 5 Regulatory Subunit Associated Protein 2 (CDK5RAP2) mutations. By embedding embryoid bodies in Matrigel following neural commitment, the authors were able to achieve interdependent brain regions following formation of functional cortical neurons ([@B92]). Further in-depth transcriptomic analysis and DNA methylome sequencing demonstrated that these cerebral organoids share a similar expression profile and epigenetic signature with their fetal counterparts, namely similar gene expression patterns for neural progenitor self-renewal, differentiation, ECM production, adhesion and migration, and thus demonstrating how organoids could be used for neurodevelopmental studies ([@B19]; [@B104]).

![Different types of cell culture formats. Differences between 2D and 3D cell culture methods are highlighted. Importantly, 3D cell culture formats have been developed to accommodate static and/or dynamic (with fluid flow/mixing) designs. The development of 3D culture methods has been prompted by scaffold-based and scaffold-free approaches that can be used for various culture methods, including microfluidic bioreactors and bioprinting. **(A)** Conventional 2D cell culture formats are illustrated along with advantages and disadvantages. Cells grow as a 2D monolayer with cell--cell contacts across a single surface. **(B)** Several scaffold-free approaches are highlighted including hanging drop and controlled aggregation methods that use gravity to assemble cells in 3D. **(C)** Scaffold based approaches include encapsulation of cells in synthetic or natural matrices that provide support to the cells and allow them to remain suspended. **(D)** The two methods of 3D cell culture have been adapted to both perfusion and static cultures in the form of microfluidic organ-on-chip platform/bioreactor platforms or as bioprinting on a surface, respectively.](fbioe-08-00692-g001){#F1}

Although Matrigel is widely used in stem cell organoid culture, its heterogenous composition poses a disadvantage to study specific spatial temporal cues that govern cell organization. As an alternative, hydrogels can be used to form 3D polymeric networks that support organoid culture under defined conditions. [@B101] developed a hyaluronic acid (HA)-based hydrogel to grow cerebral organoids and avoid matrix variability. Hydrogels can also be functionalized with ECM proteins, such as collagen to mimic a defined cell microenvironment ([@B153]; [@B120]; [@B101]). In addition, soft-lithography, including microcontact printing, has been used to promote cell aggregation in highly organized 3D structures ([@B133]; [@B11]; [@B49]; [@B50]).

Polysaccharides like alginate have also been shown to support growth of hiPSC-derived and patient-specific organoids, rendering similar phenotypic traits comparable to Matrigel-grown organoids ([@B103]; [@B162]; [@B18]; [@B21]). Osteogenesis and angiogenesis could be achieved in an artificial sphere-shaped organoid using mesenchymal stem cells (MSCs) and human umbilical vain endothelial cells (HUVECs) ([@B172]). Such an approach may provide a functional delivery method of osteogenic factors for bone injury, which could be personalized by using patient-specific MSC or hiPSC. Other studies using alginate-based matrices include hiPSC-derived organoids such as to mimic a vascularized-myocardium on-chip ([@B171]) as well as a 3D patient-specific lung organoid to study idiopathic pulmonary fibrosis ([@B162]).

The same principle of tethering ECM components to biopolymers can also be applied to synthetic hydrogels like polyethylene glycol (PEG). [@B62] compared different designs of PEG-based defined matrices for intestinal stem cell organoid culture. PEGs were functionalized with HA, fibronectin, collagen or RGD peptide domain responsible for cellular adhesion to the ECM matrix, and examined resulting organoids *in vitro* ([@B62]). Interestingly, there have been studies that do not include ECM components in matrix design. [@B30] developed a four-armed PEG-macromer that was used to generate hPSC-dervied lung organoids with *in vivo*-like epithelial morphology. Likewise, [@B20] demonstrated that only PEG-crosslinked amikacin hydrogel was sufficient to induce a 3D self-organization of hESC-derived pancreatic cells, with further phenotypic maturation achieved upon addition HUVECs that promoted endocrine-like function.

Although the aforementioned studies do not introduce ECM components in matrix design, it should be noted that such components are still produced within the organoid. Indeed, this is the premise for scaffold-free methods. In such cases, cells can be cultured in small droplets of media (hanging drop method) and forced to aggregate based solely on gravitational force ([@B73]). This approach has been used widely to establish 3D models for cardiac diseases ([@B48]; [@B10]) and cancer ([@B3]; [@B46]). Additional scaffold-free approaches include the formation of free-floating aggregates in exclusively non-adherent culture conditions ([@B47]; [@B125]; [@B13]; [@B168]), as well as the controlled aggregation of cells using microwells fabricated through soft-lithography techniques ([@B158]; [@B5]; [@B31]; [@B112]). Microfluidic devices have been employed to achieve physiologically relevant 3D organoid models. Herein, different groups have established protocols to study neural tube formation *in vitro* ([@B34]), perform drug screening using hepatic organoids ([@B117]; [@B7]; [@B122]) and to develop 3D cardiac models ([@B144]; [@B36]).

Crispr Cas9 Systems {#S3}
===================

The CRISPR Cas9 system is made up of two components - a Cas9 nuclease and a short guide RNA (sgRNA) ([@B28]). Discovered as a part of the bacterial adaptive immune system, CRISPR Cas9 has been used widely to both knock out and regulate gene expression in now many cell types ([@B106]; [@B128]; [@B130]). A CRISPR knockout can be accomplished by designing a 20-nucleotide (nt) sgRNA sequence that can bind to a specific gene of interest through complementary base pairing ([@B130]; [@B139]). Upon successful target identification, the Cas9 nuclease induces a double strand break (DSB) in the presence of a three-nucleotide protospacer adjacent motif (PAM).

The DSB causes the cells to repair the break by inserting and deleting random sequences at the site of the break through an error prone repair mechanism called Non-Homologous End Joining (NHEJ) ([@B142]). This process is often exploited when the CRISPR Cas9 system is used in stem cells to create different disease-specific models ([@B100]; [@B95]; [@B151]). In addition, through homology dependent repair (HDR) a specific gene or sequence can be inserted at the site of the break if that sequence is simultaneously introduced to the cell with the CRISPR Cas9 system ([@B170]). While this process occurs less frequently than NHEJ, HDR has been leveraged to generate multiple distinct reporter cell lines with applications in screening and cell modeling ([@B109]; [@B147]; [@B165]; [@B173]; [@B170]). Two genomic editing tools were commonly used before CRISPR Cas9 - Zinc Finger Nucleases (ZFN) and Transcription Activator Like Effector Nucleases ([@B68]). These tools use complex nuclease design methods depending on the region to be targeted or they employ difficult cloning tools. Conversely, CRISPR Cas9 requires the design of just a 20-nt RNA sequence to ensure targeted genome editing ([@B72]). In addition, Cas9-mediated editing can introduce mutations at specific sites ([@B75]; [@B129]; [@B27]).

Gene regulation with CRISPR Cas9 can be accomplished by using CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) ([@B24]; [@B43]; [@B79]). In CRISPRi/a, several point mutations in the nuclease domain create a catalytically dead dCas9 ([@B94]; [@B67]). The dCas9 is still able complex with the sgRNA but no longer has any cleavage activity. The complex, therefore, binds to the DNA target without causing a DSB it at the specific site ([@B128]). In the case of CRISPRi, the sgRNA-dCas9 complex blocks the action of RNA polymerase and prevents transcription elongation ([@B107]). The efficiency of repression can be improved by further modification of the dCas9 to include a repressive domain, such as the Kruppel-associated box (KRAB) ([@B61]; [@B82]; [@B59]). For gene upregulation, dCas9 can be fused to activator domains and targeted to promoter/enhancer regions of genes ([@B38]). Several systems have been generated, including direct dCas9 fusions (e.g., VPR, a tripartite fusion of VP64, p65, and Rta) ([@B25]), utilizing a protein scaffold (e.g., SunTag system) ([@B124]), and incorporating an RNA scaffold \[e.g., Synergistic Activation Mediator complex (SAM)\] ([@B88]).

As a result of the growing success of genome regulation with CRISPRi and CRISPRa, the nuclease null version of Cas9 has been used in other applications including single nucleotide editing, chromatin modifications, imaging genomic sequences, evaluating regulatory sequences, among others ([@B26]; [@B4]; [@B155]; [@B58]; [@B152]) ([Figure 2](#F2){ref-type="fig"}). The earliest example of single base editing was carried out with dCas9 coupled to an apolipoprotein B mRNA editing enzyme, a catalytic peptide where a cytosine group was converted to uracil or thymine ([@B87]). Other base editors that can carry out adenine--thymine base pair changes to cytosine-guanine base pairs have also been developed ([@B58]). Such tools are useful from a therapeutic standpoint as they have the ability to be used to correct diseases that arise from single base pair changes in a gene therapy strategy. For a more thorough assessment of the different applications of dCas9-mediated control of cells, readers are referred to the review by [@B126].

![Applications of CRISPR Cas9. CRISPR Cas9 can be used to introduce genetic mutations by causing a double strand break (DSB). The nuclease-null version, dCas9, can be coupled to effector domains to activate or repress gene expression or can be coupled to fluorescent proteins for imaging of genomic elements. CRISPR Cas9 knockout and effector tools have been used for screening applications. **(A)** Standard CRISPR Cas9 genome editing through introduction of mutations can be used to study the role of several genes during differentiation or to create disease models for enhanced mechanistic understanding or drug screening. **(B)** Application of dCas9 to genomic regulation in the form of CRISPRi/a can be used to study both differentiation and cellular function. **(C)** Large scale genomic screens using CRISPR Cas9/dCas9 can be used to identify essential genes for several cellular processes through gain or loss of function studies. Such screens enable identification of candidate genes from a pool for further investigation. **(D)** Coupling dCas9 to fluorescent domains, like GFP, enable facile visualization of specific regions of the genome and temporal tracking.](fbioe-08-00692-g002){#F2}

In addition to these varied applications, CRISPR Cas9 has been adapted to genome wide screens like those carried out with RNA interference (RNAi) ([@B113]; [@B149]). As a result, they have been adapted to identify genes involved in conferring cell viability, drug resistance and promoting differentiation or self-renewal in stem cells ([@B140]; [@B77]). Such screens are typically performed in a pooled or an array-based format ([@B156]). The former method delivers all of the sgRNAs simultaneously and requires a selection post-introduction to isolate the cells that take up a specific sgRNA, while the latter delivers one sgRNA to one group of cells, thereby removing the need for selection ([@B149]). While there have been several reports that have employed genome wide pooled screens in stem cells in 2D formats, more recent studies have used these tools to study 3D organoids that encapsulate adult stem cells ([@B111]; [@B132]).

There are three ways to incorporate CRISPR Cas9 to study stem cells in 3D organoids. One method relies on genome modification of cells prior to their encapsulation in a matrix ([@B95]), while a second method involves introduction of a Cas9-sgRNA complex in organoids dissociated into single cells during infection and subsequent reformation of 3D structures ([@B121]). The third method involves direction delivery of Cas9 and sgRNA to the organoids without generating single cells ([@B110]). Of the three methods, the first two have been employed more widely due to difficulties associated with efficient gene delivery through 3D matrices ([@B93]). Since hydrogel matrices can often pose diffusional limitations to highly charged, relatively large non-viral gene delivery vectors, limitations remain in using CRISPR Cas9 to study organoids. Nevertheless, viral vectors are being employed together with nucleofection and electroporation to deliver genes to spheroid and organoid cultures ([@B110]; [@B12]; [@B57]). The development of novel gene delivery tools can expand our ability to carry out direct genomic editing of organoids.

Development of Disease Models Using Crispr Cas9 Knockout {#S4}
========================================================

The use of hiPSCs/hESCs for the development of human disease models is built on their ability to adopt essentially any cellular fate through differentiation. Therefore, hiPSCs/hESCs may serve as an unlimited source of disease relevant cells. These cells can then be used to better understand pathologies associated with specific diseases, as well as to search for potential treatment options. Several diseases including Parkinson's disease, amyotrophic lateral sclerosis (ALS), cystic fibrosis, cardiac fibrosis, polycystic kidney disease, among others, have been studied using hiPSCs/hESCs and other cell types ([@B71]; [@B55]; [@B127]; [@B96]; [@B150]). Genome editing using CRISPR-Cas9 in 3D organoids has been used for such disease modeling, either by modifying a parental undifferentiated stem cell line or by rescuing a gene that is involved in causing the disease phenotype ([Figure 3](#F3){ref-type="fig"}).

![Generation of disease models using CRISPR Cas9 knockout tools. Models can be generated by either knocking out a healthy version of a gene in cells or by rescuing a defective gene. **(A)** A healthy person's somatic cells are reprogrammed into a pluripotent stage. Specific disease causing mutations can be introduced to genes in these cells by either knocking in a defective genes or introducing INDELS through genome editing. **(B)** Alternatively, a diseased patient's cells can be isolated and reprogrammed into a pluripotent stage. These cells can be then fixed by correcting the mutation by knocking in a functional version of the gene or introducing healthy version at the adeno-associated virus integration site (AAVS1).](fbioe-08-00692-g003){#F3}

Generating Cells With Disease-Carrying Mutations {#S4.SS1}
------------------------------------------------

To introduce disease mutations and potentially mimic disease phenotypes, CRISPR Cas9 has been used to knock out various genes in healthy stem cells. Several types of organoids, including those consisting of brain ([@B83]), lung ([@B151]), kidney ([@B53]), intestinal ([@B78]), and other stem cells have been generated from these knockout stem cell types to better model *in vivo* environments. A majority of studies have focused on better understanding brain disorders with cerebral organoids and hence this is a major focus of this section.

A major advantage of genome engineering is the ability to study diseases in experimentally relevant human cell types. For example, patient-specific iPSCs were used to create a disease model of frontotemporal dementia (FTD), wherein CRISPR Cas9 was used to introduce mutations in the MAPT gene ([@B115]). There were several changes noted in generated neurons due to the mutation including lower phosphorylation levels in the tau protein, which resulted in structural reorganization of the microtubules as well as reduced numbers of mitochondria in the cells.

In addition, the specificity of gene targeting with CRISPR/Cas9 enabled the generation of similar mutations that are found in patients carrying a specific disease. This was shown with a cerebral organoid disease model for GM1 ganglioside storage disease that results in extensive buildup of a glycosphingolipid ganglioside due to a non-functional cellular β-galactosidase ([@B157]). Human iPSCs were used to prepare isogenic cell lines with the disease carrying mutations in specific exons where most mutations for the disease were recorded in patients ([@B95]). Following gene editing, increased levels of GM1 were observed in mutated organoids over non-mutated controls leading to GM1 ganglioside storage disease phenotypes recapitulated in gene-edited organoids.

Phenotypes of major neurodegenerative diseases like Alzheimer's disease and Parkinson's disease can be recapitulated in a 3D organoid environment by CRISPR Cas9-mediated editing ([@B100]; [@B83]). For Alzheimer's disease, cerebral organoids were prepared from hiPSCs that harbored the disease causing variant of apolipoprotein E (APOE4), which was found to show ∼2 fold increase in the accumulation of amyloid β than control organoids that had the APOE3 gene variant ([@B100]). For Parkinson's disease, [@B83] generated a single point base mutation to model a specific form of the disease, the LRRK2-associated sporadic disease. Cerebral organoids generated from mutated hiPSCs were then found to yield several different phenotypes that were observed with Parkinson's disease and also used to identify a disease relevant gene encoding for the thioredoxin interacting protein (TXNIP) ([@B83]). They further showed that the gene edited dopaminergic neurons showed enhanced cytotoxicity as indicated by a ∼20% increase in caspase-3 activity. These studies are especially relevant since CRISPR-edited iPSCs have the ability to generate a near limitless supply of cells for studying these diseases as opposed to patient derived adult cells, which are far more finite.

Structural conditions like macrocephaly, microcephaly and cell migration can be studied more accurately with 3D organoid models incorporating CRISPR induced mutations than with animal models. To this end, [@B97] generated a Phosphatase and Tensin Homolog (PTEN) gene knockout model to activate PTEN-AKT and cause 3D organoids to display a macrocephaly phenotype. The authors were thus able to address several interspecies differences in this pathway including stark differences in the expansion and folding mechanism of human organoids when compared to mice. Therefore, CRISPR Cas9 can be used to address some of the issues associated with translating results between multiple species by enabling study in the more relevant human cell lines.

Finally, genome modification to multipotent stem cell types, including neural stem cells (NSCs), have been used to model diseases like cancer in cerebral organoids ([@B12]). Direct introduction of CRISPR Cas9 for mutating several tumor suppressor genes in 3D embryoid bodies was performed at the NSC stage to identify key genes, which when mutated, resulted in abnormal growth associated with various cancers. Mutations in certain genes resulted in rapid proliferation of the organoids in culture. Further investigation revealed mRNA changes in the expression of several genes between mutated cancerous organoids and non-cancerous organoids. Such disease models have yielded a wealth of information on how cells change with tumorigenesis and how this information could be leveraged to generate better potential drug candidates to reduce tumorigenesis ([@B105]).

A cohort study conducted by [@B159] is an interesting example of how CRISPR-engineered organoids are useful in determining genotypic-phenotypic associations. This study provided insights of the mechanism by which mutations in the diacylglycerol-acyltransferase 1 (DGAT1) gene is associated to congenital intestinal disorders. The authors used CRISPR-Cas9 to generate knockouts of the DGAT1 gene on cells obtained from duodenal biopsies of three healthy individuals and compared lipid production to patient-derived organoids that contain the mutations leading to similar results.

Kidney organoids have been studied in detail using CRISPR Cas9-edited hiPSCs to model renal diseases. A knockout of phosphoinositide dependent kinase 1 (PDK1) and phosphoinositide dependent kinase 2 (PDK2) used to model disease pathology of polycystic kidney disease showed enhanced cyst formation when cells were grown into kidney organoids ([@B53]). Some of the lesser-studied organoid models with CRISPR Cas9 include lung organoid models where only one group has done major work to study a lung disease in 3D format using CRISPR Cas9 ([@B151]). Nevertheless, this shows that Cas9 mediated knockout can be used to study multiple different organoid systems. A list of studies that have utilized CRISPR Cas9 to introduce disease causing mutations in stem cells for further study in 3D cell cultures are given in [Table 1](#T1){ref-type="table"}.

###### 

Examples of CRISPR Cas9 to introduce disease causing mutations.

  **Edited cell type**   **Type of organoid**   **Gene knockout**                 **Disease modeled**                                **Patient derived (Y/N)**   **References**
  ---------------------- ---------------------- --------------------------------- -------------------------------------------------- --------------------------- -----------------------------------
  iPSC                   Cerebral               MAPT                              Frontotemporal dementia                            Y                           [@B115]
  iPSC                   Cerebral               GLB1                              GM1 ganglioside storage disease                    N                           [@B95]
  iPSC                   Cerebral               RS1                               X-linked juvenile retinoschisis                    Y                           [@B74]
  iPSC                   Cerebral               AXL                               Zika virus                                         N                           [@B161]
  ESC                    Cerebral               LIS1                              Lissencephaly                                      N                           [@B81]
  iPSC                   Cerebral               APOE3                             Alzheimer's disease                                N                           [@B100]
  iPSC                   Cerebral               LRRK2                             Parkinson's disease                                N                           [@B83]
  ESC                    Cerebral               PTEN                              Zika virus                                         N                           [@B97]
  NSC derived from ESC   Cerebral               Multiple tumor suppressor genes   Brain tumor                                        N                           [@B12]
  iPSC                   Cerebral               TSC1, TSC2                        Tuberous sclerosis                                 N                           [@B14]
  Organoids from hESC    Cerebral               TP53                              Glioblastoma                                       N                           [@B119]
  iPSC                   Cerebral               DCHS1, FAT4                       Neuronal heterotopia                               Y                           [@B86]
  iPSC                   Cerebral               CLN3                              Neuronal ceroid lipofuscinosis                     N                           [@B63]
  iPSC                   Cerebral               CDH8                              Schizoaffective disorder                           Y                           [@B160]
  iPSC/ESC               Kidney                 PODXL                             Podocyte development                               N                           [@B53]; [@B84]
  ESC                    Kidney                 PKD1, PKD2                        Polycystic kidney disease                          N                           [@B53]
  ESC                    Intestinal             STAT3                             Intestinal organoid maturation                     N                           [@B78]
  ISC                    Intestinal             APC, TP53, KRAS, SMAD4            Colorectal cancer                                  Y                           [@B42]; [@B110]; [@B121]; [@B134]
  ISC                    Intestinal             MLH1, NTLH1                       Colorectal cancer                                  Y                           [@B41]
  ESC                    Lung                   HPS2, HPS4, HPS8                  Hermansky-Pudlak syndrome interstitial pneumonia   N                           [@B151]
  ISC                    Intestinal             DGAT1                             Congenital diarrheal disorder                      Y                           [@B159]

Genome wide pooled screening tools have only recently been employed to study stem cells in 3D organoid and spheroid cultures ([@B111]; [@B132]). [@B111] and [@B132] used adult intestinal stem cells and cancer stem cells to identify genes that prevent cells from responding to transforming growth factor-β (TGF-β) signaling. As a result, pooled CRISPR screens can effectively generate large amounts of data on the roles of several genes simultaneously. These experiments show the possibility of conducting screening experiments with stem cells and can now be expanded for use in studying pluripotent and multipotent stem cells. While a few experiments have been performed in 2D, to our knowledge there are no reported examples in 3D.

Animal models have been useful in helping us study disease progression as well to evaluate treatment options. Xenotransplantation of CRISPR engineered organoids are particularly useful as they do not require complex methods needed to generate mice carrying specific mutations ([@B134]). Modified tissue xenografts have been studied extensively with human and rodent adult intestinal stem cells ([@B42]; [@B110]; [@B121]; [@B134]). These genome-edited organoids were used to evaluate tumor progression during cancer development ([@B42]; [@B121]). In addition, such studies enable the development of gene expression profiles in tumors and essential mutations needed for cancer metastases ([@B110]; [@B134]).

Generating Healthy Cells With Gene Rescue {#S4.SS2}
-----------------------------------------

CRISPR Cas9 has been used to perform gene rescue and better understand disease pathologies. One of the earliest examples of using CRISPR Cas9 to repair a mutant gene was done using cystic fibrosis transmembrane conductor receptor (CFTR) ([@B138]). Adult intestinal stem cells were transfected with Cas9, sgRNA, and wild type CFTR genes to allow for homologous recombination of the corrected gene following which a forskolin based swelling assay was carried out ([@B138]). When these cells were grown into intestinal organoids, the corrected organoids displayed increased swelling in comparison to the diseased organoids (non-mutant) when they were subjected to a forskolin based assay. Forskolin swelling assay is commonly used to study cystic fibrosis since diseased cells carry a non-functional CFTR channel that prevents swelling of cells upon exposure to forskolin since the channel is unable to transport chloride ions into the cells ([@B15]).

Disease correction has also been carried out with CRISPR Cas9 in hiPSCs. This has important implications in regenerative medicine, as such genome editing tools may be used therapeutically. Diseased or mutant gene alleles can be modified into "healthy" versions in reprogrammed hiPSCs, which subsequently can be differentiated to any target cell type that can serve as a replacement to the diseased tissue. Some examples are summarized in [Table 2](#T2){ref-type="table"}. Gene rescue in hiPSCs has been used to generate multiple organoid types including cerebral ([@B2]), intestinal ([@B60]), kidney ([@B52]), and retinal ([@B35]) organoids.

###### 

CRISPR Cas9 examples to rescue genes.

  **Cell type**                         **Organoid**   **Gene rescued**        **Disease studied**                   **Patient derived (Y/N)**   **References**
  ------------------------------------- -------------- ----------------------- ------------------------------------- --------------------------- ----------------
  ISC                                   Intestinal     CFTR                    Cystic fibrosis                       Y                           [@B138]
  iPSC                                  Intestinal     DKC1                    Dyskeratosis congenita                Y                           [@B163]
  ISC                                   Intestinal     CFTR                    Cystic fibrosis                       Y                           [@B60]
  iPSC                                  Cerebral       HEXB                    Sandhoff disease                      Y                           [@B2]
  iPSC                                  Cerebral       CNTNAP2                 Autism spectrum disorder              N                           [@B33]
  iPSC                                  Kidney         IFT140                  Nephronophthisis related ciliopathy   Y                           [@B52]
  iPSC                                  Retinal        RPGR                    Retinitis pigmentosa                  Y                           [@B35]
  iPSC                                  Cerebral       LIS1, YWHAE (14.3.3e)   Miller-Dieker syndrome                N                           [@B76]
  Hepatocytes from fetal human livers   Hepatic        TP53                    N/A                                   N                           [@B6]

In another example of gene rescue, a dyskeratosis congenital (DC) intestinal organoid model was used to identify the Wnt signaling pathway as a potential therapeutic target for the disease ([@B163]). CRISPR Cas9 corrected DC cells that were isolated from patients showed higher expression of several genes regulated by Wnt signaling. Specifically, Ascl2, a known regulator of intestinal stem cell development ([@B137]) showed differential expression between diseased and corrected organoids ([@B163]). The success in using intestinal organoids to model diseases has also resulted in newer CRISPR technologies including adenine base editing used to correct disease causing mutations with a large set of patient derived cells ([@B60]). Four types of patient derived cells were corrected using a Cas9 base pair editor that allows for switching an adenine--thymine (A--T) base pair to cytidine--guanine base pair (C--G). This work is especially important since a large number disease causing mutations are due to a base pair substitution event (C--G base pair change to A--T) ([@B58]). Furthermore, the ability to utilize these base-editing tools with multiple different patient samples shows the versatility of CRISPR Cas9 for therapeutic purposes.

Normal cell function was restored in several other organoid models including cerebral ([@B2]), kidney ([@B52]), and retinal ([@B35]) organoids using CRISPR Cas9. For example, CRISPR Cas9 was used to correct a mutation and rescue a gene involved in causing Sandhoff disease (a lysosomal storage disorder) from patient derived cells ([@B2]). Specifically, gene edited organoids showed reduced proliferation and did not show developmental abnormalities that were found with the diseased organoids. With kidney organoids, the intraflagellar transport 140 (IFT140) gene was rescued in cells derived from a patient who had nephronophthisis (NPHP)-related ciliopathy (NPHP-RC) ([@B52]). There were several structural differences as well as gene expression differences in the organoids, especially in genes involved in the creation of apicobasal polarity, the formation of cell--cell junctions and in the assembly of dynein motor proteins. Finally, several phenotypic changes associated with retinitis pigmentosa were corrected with the use of CRISPR Cas9 mediated editing of retinitis pigmentosa 3 GTPase regulator (RPGR) in 3D retinal organoid model ([@B35]). The authors were able to reverse several disease-related symptoms including loss of photoreceptors and ciliopathy in the patient derived cells by correcting a frameshift mutation in the RPGR gene.

Alternatively, the safe harbor AAVS1 locus of patient-derived hiPSC cells can be used as a site to introduce the Cas9 encoding gene and a functional version of the gene to be rescued ([@B118]; [@B76]). For example, [@B76] transfected Miller-Dieker syndrome patient-derived hiPSCs with plasmids encoding Cas9 and two other proteins (LIS1 and 14.3.3e) to partially rescue the lissencephaly phenotype. They further showed that the neuroepithelial loops that were generated upon differentiation showed increased loop diameter and apical membrane length in organoids (∼50 μm increase ∼200 μm increase, respectively, for LIS1 rescue and ∼75 and ∼100 μm increase, respectively, for 14.3.3e rescue).

Most of the aforementioned studies described in this section resort to HDR to introduce the gene mutation at a specific site, as this approach relies on homology arms present in the donor plasmid. Nevertheless, NHEJ-mediated mechanisms potentially could be used to generate knock-in functional genes ([@B6]). As a proof-of-concept, [@B6] developed a CRISPR-Cas9-mediated homology-independent organoid transgenesis method, coined CRISPR-HOT, to mediate precise gene insertion in different types of organoids, namely liver-, and intestinal-derived tissues. When compared to HDR, a 10-fold higher efficiency was achieved with precise in-frame insertions of a gene encoding a fluorescent protein used for C-terminal tagging. Although this approach is a useful tool for reporter cell line generation and labeling cellular structures, it can also be multiplexed with gene knock-out studies, as demonstrated by the loss of TP53 gene in hepatic organoids, resulting in an aberrant mitotic spindle behavior and disorganization of microtubules. Thus, CRISPR-HOT may be useful in generating myriad disease models ([@B6]).

Conclusion and Future Perspectives {#S5}
==================================

Although remarkable advancement in 3D model development has been achieved, there remain important questions to be answered. Recently, several vascularization processes have been incorporated into 3D models ([@B80]; [@B123]; [@B89]; [@B164]). Two such methods include bioprinting encapsulated cells in synthetic hydrogels resulting in perfusable networks through two photon polymerization ([@B123]) or co-culturing organoids with endothelial cells ([@B80]; [@B89]). For a detailed summary of methods used to generate vascular tissue, readers are referred to the review by [@B66]. Despite progress made toward incorporating vascular structures in 3D organoid models, there are few studies on the use of such tools in understanding disease progression with CRISPR-modified organoids. Combining vascularization with specific disease-causing mutations in 3D may help yield more personalized patient models.

In addition, while the majority of this review has focused on using CRISPR Cas9 knockout tools for studying diseases, some groups have also started using CRISPRi/a for similar studies ([@B69]). This is especially important because CRISPRi/a allows for temporary modifications to gene expression through inducible promoters as opposed to the more permanent gene knockout experiments. Such experiments can rapidly simplify the process of studying diseases in 3D organoids by removing the need for creating multiple different cell lines with and without mutations and also allows for dynamic control of disease phenotypes with a single population of cells. However, most such studies have been carried out in 2D cell formats. As an example, [@B70] demonstrated that the VPR (a tripartite fusion of VP64, p65, and Rta) domain coupled to dCas9 could be used to induce activation of endogenously expressed SCNA gene in healthy neurons, which led to an eightfold increase of the alpha-synuclein protein associated with Parkinson's disease ([@B70]). In parallel, the KRAB domain was used to reduce the transcriptional expression of the same protein in diseased-phenotypic neurons that led to a 40% reduction relative to a non-edited Parkinsonian hiPSC-derived neuron ([@B69]). Despite significant improvements in understanding the role of the SCNA gene, it is unclear how it would influence the 3D structure of the organoid, or how the specific microenvironmental cues could potentially influence epigenomic regulation of the edited gene ([@B102]). Modulating gene expression of SCNA or any other gene of interest in organoids and spheroids with CRISPRi/a tools can help address several of these unknowns.

Another important aspect that requires further study in this area is the use of artificial intelligence and machine learning tools coupled to CRISPR-engineered organoids. For instance, groundbreaking work developed by [@B99] demonstrate that machine learning algorithms could empirically determine and control the spatial self-organization of multicellular patterns without the need for micropatterning technique or other extrinsic patterning methods. Other studies demonstrated that trained machined learning models could be used to identify possible mutations (indels) generated by CRISPR genome editing and repair ([@B1]). This could be further used to improve disease models ([@B143]).

Finally, novel CRISPR Cas tools that utilize other Cas proteins including Cas12a and Cas13 and their catalytically dead counterparts, have been discovered specifically for nucleic acid detection and protein detection ([@B64]; [@B8]; [@B32]). Of these two, Cas13 has the ability to target RNA sequences ([@B65]). Hence it has been used for several applications including detecting RNA viruses, downregulating genes by targeting RNA transcripts, imaging RNA sequences, etc. ([@B65]; [@B54]). RNA imaging is a powerful application because it potentially can be used as an alternative for fluorescence *in situ* hybridization (FISH) in live cells ([@B167]). Using this tool to study 3D organoids can allow for rapid real-time tracking of several genes that may not just be involved in causing diseases but also in elucidating healthy development and differentiation of stem cells.

In this review we addressed several examples in the use of CRISPR Cas9 tools to repair defective genes in diseased organoids. The ultimate goal of such studies is to aid in the development of emerging treatments that employ gene therapy, chimeric antigen receptor T cell therapy or cell therapy. This requires *in vivo* delivery of the Cas9-sgRNA complex, and examples of this have been shown for rodent disease models ([@B37]; [@B9]; [@B56]). While several therapeutic challenges remain, these animal models suggest that it may be possible to target defective genes in humans. Therefore, addressing these areas, particularly in patient-derived hiPSCs that are differentiated into specific adult stem cells and terminal progeny represents an opportunity to bring the "bedside" to the "bench" and advance therapeutic options for patients.

Author Contributions {#S6}
====================

SG and AR wrote the manuscript along with JD. JD edited the manuscript. All authors contributed to the article and approved the submitted version.

Conflict of Interest {#conf1}
====================

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

**Funding.** This work was partly funded under a Project Award Agreement from the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) and financial assistance award 70NANB17H002 from the U.S. Department of Commerce, National Institute of Standards and Technology. The authors acknowledge financial support from Fundação para a Ciência e a Tecnologia (FCT), Portugal through iBB -- Institute for Bioengineering and Biosciences (UID/BIO/04565/2013) and from Programa Operacional Regional de Lisboa 2020 (Project N. 007317). AR acknowledges FCT for financial support, for the scholarship with the reference (SFRH/BD/135524/2018).

[^1]: Edited by: Stephanie Michelle Willerth, University of Victoria, Canada

[^2]: Reviewed by: Jatin Roper, Koch Institute for Integrative Cancer Research at MIT, United States; Jennifer Patterson, KU Leuven, Belgium

[^3]: ^†^These authors have contributed equally to this work

[^4]: This article was submitted to Biomaterials, a section of the journal Frontiers in Bioengineering and Biotechnology
