Human Genetics

, Volume 137, Issue 9, pp 679–688 | Cite as

Application of CRISPR/Cas9 technologies combined with iPSCs in the study and treatment of retinal degenerative diseases

  • Bincui Cai
  • Shuo Sun
  • Zhiqing Li
  • Xiaomin Zhang
  • Yifeng Ke
  • Jin Yang
  • Xiaorong Li


Retinal degeneration diseases, such as age-related macular degeneration and retinitis pigmentosa, affect millions of people worldwide and are major causes of irreversible blindness. Effective treatments for retinal degeneration, including drug therapy, gene augmentation or transplantation approaches, have been widely investigated. Nevertheless, more research should be dedicated to therapeutic methods to improve future clinical treatments. Recently, with the rapid development of genome-editing technology, gene therapy has become a potentially effective treatment for retinal degeneration diseases. A clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been developed as a powerful genome-editing tool in ophthalmic studies. The CRISPR/Cas9 system has been widely applied in basic research to develop animal models and gene therapies in vivo. With the ability to self-renew and the potential to differentiate into different types of cells, induced pluripotent stem cells (iPSCs) have already been used as a promising tool for understanding disease pathophysiology and evaluating the effect of drug and gene therapeutics. iPSCs are also a cell source for autologous transplantation. In this review, we compared genome-editing strategies and highlighted the advantages and concerns of the CRISPR/Cas9 system. Moreover, the latest progress and applications of the CRISPR/Cas9 system and its combination with iPSCs for the treatment of retinal degenerative diseases are summarized.


Retinal degeneration diseases target photoreceptors or the adjacent retinal pigment epithelium (RPE). Degeneration, necrosis and apoptosis of light-sensing photoreceptors are the common pathophysiologic processes that cause severe or complete loss of vision (Huang et al. 2011). For decades, scientists have tested a variety of methods to prevent vision loss or restore the vision of patients affected by retinal degeneration, such as gene therapy, growth factor therapy, retinal transplantation, artificial retina implantation, and so on. Genome-editing technology has quickly developed in recent years. Four common nuclease-based platforms are as follows: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and most recently, clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) systems (Yanik et al. 2016). Previous approaches have been hampered by inaccuracy and high rates of off-target effects, while the CRISPR/Cas9 system is a more accurate and efficient method to edit the human genome (Chrenek et al. 2016). Along with the extensive development of CRISPR/Cas9 research, numerous breakthroughs have been made in improving the specificity and efficacy of CRISPR/Cas9-mediated genome editing. Several related innovated software programs have been developed for predicting off-target cleavage sites that can be helpful for reducing off-target effects (Stemmer et al. 2015; Zhu et al. 2016). The“anti-CRISPR” protein AcrIIA4, which is known as the most potent Spy Cas9 inhibitor in human cells and acts as a DNA mimic to block protospacer adjacent motif (PAM) recognition, allows on-target Cas9-mediated gene editing while reducing off-target edits. Shin et al. predicted that Cas9 inhibitors could be widely used for allele-specific therapeutic editing in which the precise control of either on- or off-target gene editing is desirable (Shin et al. 2017). In addition, more efficient and appropriate gene delivery systems have been developed (Nelson and Gersbach 2016; Yanik et al. 2017). As an efficient genome-editing tool, the CRISPR/Cas9 technique has been widely used in retinal degenerative disease modeling and gene therapy exploration. Moreover, target gene activating (TGA) CRISPR/Cas9, a new type of CRISPR system known as CRISPRa, could be used to turn on dormant genes instead of snipping them for treating a variety of diseases, which is more safe and manageable. This is a robust system for in vivo activation of target genes through trans-epigenetic remodeling. In this system, modified single guide RNAs were used to target loci through recruitment of Cas9 and transcriptional activation complexes. Although CRISPRa has not been used in ocular diseases, it presents a new research tendency (Liao et al. 2017).

With the characteristics of pluripotency and autology, induced pluripotent stem cells (iPSCs) are immunologically safer and avoid ethical controversies. Additionally, the various somatic cells that are differentiated from patient-specific iPSCs are ideal tools for disease modeling and transplantation research (Wiley et al. 2015). Combined with iPSC technologies, the CRISPR/Cas9 technology shows great potential for use in gene and cell therapy in vivo. Nonetheless, several challenges must be addressed before moving CRISPR/Cas9 therapies to the ophthalmic clinic (Fig. 1).

Fig. 1

Structure and applications of the CRISPR/Cas9 system in retinal degeneration diseases

Varieties of genome-editing methods and targeted nucleases

Targeted genome-editing technology can be applied to ophthalmic genetic diseases by correcting the disease-causing mutations or inserting protective mutations, depending on the capacity of the cells to repair DNA double-strand breaks (DSBs) and highly specific endonucleases (Cox et al. 2015). The field of genome editing is based on the discovery that targeted DNA DSBs can stimulate endogenous cellular repair machinery. DSBs are repaired in one of three ways: (a) homology-directed repair (HDR) by using the sister chromatid as template DNA; (b) nonhomologous end joining (NHEJ), in which DNA ends are stuck without any homology; and (c) microhomology-mediated end joining (MMEJ), in which DNA ends are brought together with a few nucleotides of homology, termed microhomology (Jasin and Haber 2016). Considering endogenous DNA repair functions, various site-specific genomic alterations can be engineered through the precise introduction of a targeted DSB, for which highly specific endonucleases are essential (Fig. 2). Four major programmable nucleases can be applied to induce site-specific DSBs: meganucleases, ZFNs, TALENs, and CRISPR-associated nuclease/Cas9 (CRISPR/Cas9) (Yanik et al. 2016). The features of these four genome engineering platforms are compared in Table 1.

Fig. 2

CRISPR/Cas9 genome-editing schematic diagram: Cas9 nuclease with sgRNA complexes can produce specific double-strand breaks (DSBs) in host DNA. The targeted DNA DSBs can stimulate endogenous cellular repair machinery. DSBs are repaired in one of three ways: a homology-directed repair (HDR) using the sister chromatid as template DNA will introduce precise genomic changes in the host’s DNA; b nonhomologous end joining (NHEJ), in which DNA ends are stuck without any homology; and c microhomology-mediated end joining (MMEJ), in which DNA ends are brought together with a few nucleotides of homology, termed microhomology. NHEJ and MMEJ most likely result in the introduction of insertions and deletions

Table 1

Comparison of features of genome engineering platforms



DNA-binding domain





Homing endonucleases (I-CreI and I-SceI enzymes)

Re-engineered homing endonucleases to target novel sequences

Large gene segment

More recombinogenic for HDR

Amenable to all standard gene delivery methods

DNA-binding and cleavage domains of homing endonucleases are difficult to separate

Difficulty in engineering proteins

Lower efficiency of identification and cut


Synthetic nucleases (FokI DNA-binding domain replaced with a zinc finger domain)

Novel proteins engineered for each DNA target site (spatial dependence of DNA binding)

Human somatic; pluripotent stem cells

The smaller size of DNA-binding domains

Spatial dependence of DNA binding

Off-target effect

Extremely toxic mutation


Engineered nucleases (artificial proteins composed of a customized sequence-specific DNA-binding domain fused to FokI DNA-binding domain)

Engineered for each DNA target site (spatial independence binding of TALEN and DNA)

Target virtually any sequence

Unlimited targeting range; increased recognition specificity

Less off-target effect

The binding of TALEN and DNA exists spatial independence

Large size and repetitive nature of TALE arrays are difficult for packaged in viral systems and in vivo delivery

Unclear off-target effect


RNA-guided nucleases (Cas9 protein)

Altered short region of the guide RNA

Multiple gene editing

High efficiency of identification and cut

No species limits

More easier to design and generate the sgRNA

Cas proteins are unstable

Off-target effect

Advantages and concerns of the CRISPR/Cas9 system


Compared with ZFNs and TALENs, CRISPR-based genome editing has some advantages in application: (1) the short guide RNAs (sgRNAs) are easier to design and quicker to generate than the protein-based DNA targeting motifs used in ZFNs and TALENs (Lim et al. 2016). The sgRNAs of the CRISPR system consist of an NGG sequence, called the PAM, downstream of a 17–20-nucleotide DNA target (Maeder et al. 2013; Mali et al. 2013). (2) CRISPR-based genome editing is more efficient with greater specificity than other genome-editing tools (Auer et al. 2014). (3) CRISPR-based genome editing is more powerful for multiplex gene editing and can introduce or knock out multiple genes simultaneously with the use of different sgRNAs (Zetsche et al. 2017). Therefore, the CRISPR/Cas9 system is a powerful and useful tool for studying polygenic disease mechanisms and establishing animal models to identify novel therapeutic targets.

Concerns and solutions

Off-target effects

When CRISPR/Cas9 targeting recognizes the target sequence, it is possible to cut several mismatched bases, resulting in DSBs in undesired locations (Fu et al. 2013) that may activate a tumor suppressor gene. It is necessary to detect and reduce off-target effects before being used for in vivo gene editing in clinical applications. Several strategies have been recently developed. Some target prediction tools, such as CCTop and CT-Finder, provide rapid and efficient identification of high-quality target sites (Stemmer et al. 2015; Zhu et al. 2016). Another high-fidelity variant harboring alterations, SpCas9-HF1, was designed to reduce nonspecific DNA contacts (Kleinstiver et al. 2016). In addition, Ran et al. described an approach that combines Cas9 nickase mutants with paired guide RNAs to introduce targeted DSBs. They concluded that this approach can reduce off-target activity in cells by 50- to 1500-fold and mediate efficient indel formation in mouse embryos, suggesting the potential utility of employing nickases to increase the specificity and safety of CRISPR/Cas9 genome editing (Ran et al. 2013). Recently, Shin et al. showed that anti-CRISPR protein AcrIIA4 binds to assembled Cas9–sgRNA complexes but not to Cas9 protein only. The timed delivery of AcrIIA4 to human cells, either as a protein or an expression plasmid, can be employed to regulate the efficacy of gene editing at multiple gene loci and reduce off-target editing (Shin et al. 2017).

Gene delivery methodologies

While low efficiency gene transfer is also a major hurdle that must be overcome for in vivo retinal genome editing, it is crucial to choose an optimal vector system. Various gene delivery systems are available to transfer genes into the retina; their characteristics are summarized in Table 2 (Nelson and Gersbach 2016). In CRISPR/Cas9-mediated gene editing in vivo, two components (the Cas9 gene and one or more gRNAs) for NHEJ or three components (Cas9, gRNAs, and a donor template) for HDR must be delivered to the target cells. Direct delivery of CRISPR/Cas9 components to retinal tissue for genome surgery in vivo is an important issue that is being studied. The transfer efficiency of plasmids to most quiescent cells in adult tissues remains low. To improve the efficiency, Suzuki et al. devised a homology-independent targeted integration (HITI) strategy based on CRISPR/Cas9 technology, allowing for robust DNA knock-in in both dividing and nondividing cells in vitro and in vivo (Suzuki et al. 2016). Young et al. found that a DNA nuclear targeting sequence (SV40 DTS) with the ability to target DNA to the nucleus can increase gene transfer in vivo. The inclusion of the SV40 sequence in plasmids will enhance nonviral gene delivery (Acland et al. 2005). Hung et al. demonstrated that genomic modification of cells in the adult retina can be readily achieved by viral-mediated delivery of CRISPR/Cas9 by using Thy1-YFP (yellow fluorescent protein) transgenic mice as a rapid quantifiable means to assess the efficacy of a CRISPR/Cas9 system for retinal modification in vivo (Jacobson et al. 1990).

Table 2

Characteristics of major gene delivery methodologies in the retina in vivo





Circular DNA molecules

Single-stranded DNA parvovirus

Retroviruses with an ssRNA genome

Capacity and vectors

Intravitreal or subretinal injection coupled with ultrasonic microbubble disruption or electroporation

Packaged into lipid small unilamellar liposome vesicles (SUVs), intravitreal or subretinal injection

Packaged into nanoparticle, intravitreal or subretinal injection (14 kB)

Low cargo capacity (4.7 kB) AAVs vector

Large trans genes up to  (8 kB) lentiviral vector

Application and advantages

The “mini-circles”—cheapest and easiest to produce

Delivering large number of plasmids into each transfected cell

Delivering large number of copies of the plasmids into each cell

Applicable to dividing and non-dividing cells

Remains episomal and shows no toxicity

Expresses packed genes faster

Gold standard for retinal gene transfer

Applicable to non-dividing cells

Promising vehicles to transfer genes into iPSCs

Transfers vectors for endonucleases and template


The method of making pores in the cell membrane can lead to death of target cells

Chemicals used to make the lipid vesicles are toxic to cells

Difficulty in remaining stable in the blood stream

Low cargo capacity is not sufficient for some nucleases

Not as safe as non-integrating vehicles

Transduction efficiency in PR is limited

Adeno-associated virus (AAV) is a small nonpathogenic dependovirus that has shown great potential for safe and long-term expression of a genetic payload in the retina and has been commonly used in studies of inherited retinal degeneration treatments in animal models (Day et al. 2014). Senís et al. reported AAV/CRISPR vectors that can be exploited for gene engineering in vivo, as exemplified in adult mouse livers, for accelerating clinical applications (Senís et al. 2015). A novel intravitreal injection procedure described by Da et al. led to reproducible AAV2/8-mediated transduction of more than 70% of the retina, providing a potential method for applying gene replacement strategies or CRISPR/Cas9 in vivo. Application of the AAV2/8 technique to the vitreous of mice leads to widespread transduction of the retina (Da et al. 2016). In conclusion, compared with these viral and nonviral modes of gene transfer, recombinant AAV continues to be a popular vector that is currently used in the eye for CRISPR/Cas9-mediated genome editing both in therapeutic and transplantation studies. Each gene transfer system has both merits and limitations; thus, cotransfection with multiple vectors containing the appropriate genetic information may be needed. Moreover, a new approach was put forward by Yanik et al. involving combined delivery of the endonuclease as protein and an AAV vector containing the template, which may be the optimal choice. Protein delivery guarantees a temporal burst of endonuclease activity, while AAV delivery increases the activity of DNA repair machinery by itself (Yanik et al. 2017).

In summary, compared with other genome-editing technologies, the CRISPR/Cas9 system is more efficient and powerful for multiplex gene editing. However, the off-target effects of the CRISPR/Cas9 system remain a controversial problem (Schaefer et al. 2017, 2018). Nonetheless, the CRISPR/Cas9 TGA system, which activates target genes in vivo without editing DNA sequences, has great potential for future applications.

Applications of the CRISPR/Cas9 system in retinal degenerative diseases

Generating disease models for pathophysiological investigations or therapeutic explorations

With no specific-species limitations, the CRISPR/Cas9 system can be widely used for genome modifications in animal disease models (Schwank et al. 2013). Some studies have used this CRISPR/Cas9 technology to create animal models for retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), retinoblastoma (Rb), cataracts, etc.

Retinitis pigmentosa (RP) is an inherited retinal disorder that is caused by genetic mutations and is marked by photoreceptor and retinal pigment epithelial cell dysfunction; it is responsible for vision loss in many people worldwide (He et al. 2015). Arno et al. used CRISPR/Cas9-mediated genome editing to produce Reep6 knock-in mice with the p.Leu135Pro RP-associated variant. The homozygous knock-in mice mimic the clinical phenotypes of RP, including progressive photoreceptor degeneration and rod photoreceptor dysfunction, providing a good animal model for basic research of RP (Arno et al. 2016). The preclinical model of RP is the “rodless” (rd1) mouse, with compound homozygous mutations in Y347X and Xmv-28. To distinguish which mutation causes retinal degeneration, Wu et al. used CRISPR/Cas9 gene editing in mice and concluded that the Y347X mutation in rd1 mice is pathogenic (Wu et al. 2016). As proved by Yin et al., the delivery of sgRNAs to the eye, targeting Ascl1a, a zebrafish retinal regeneration gene, leads to impaired damage-induced photoreceptor regeneration (Chavala et al. 2005). LCA is clinically characterized by severe and early vision loss, sensory nystagmus, amaurotic pupils, fundus changes and minimal or absent electrical signals on electroretinograms (ERGs) and is associated with KCNJ13 gene mutations. In a previous study, the CRISPR/Cas9 system was used to generate Kcnj13 mutant mice by zygote injection to verify the pathogenic role of human KCNJ13, further demonstrating that CRISPR/Cas9 engineered mosaicism can be used to rapidly test specific gene function in vivo (Zhong et al. 2015). A mosaic model for LCA can be quickly created by the CRISPR/Cas9 system in which mutant and wild-type cells are juxtaposed, allowing the study of KCNJ13 in pathogenesis and overcoming the problem of homozygous lethality.

In addition, CRISPR/Cas9 technology can be used to generate animal models not only of retinal degenerative diseases, but also of other ocular diseases. Retinoblastoma (Rb) is a pediatric eye tumor caused by biallelic inactivation of the Retinoblastoma 1 (RB1) gene. Naert et al. found rapid retinoblastoma animal models in Xenopus tropicalis, providing unique possibilities for fast elucidation of novel drug targets by triple multiplex CRISPR/Cas9 gRNA injections (RB1 + RBL1 + modifier gene) (Thomas et al. 2016). Cataract is a main cause of vision impairment worldwide; however, surgical treatment can restore vision in cataract patients. Through coinjection of Cas9/sgRNA mRNA into rabbit zygotes, a cataract model with GJA8 gene knockout was developed by Lin et al.; this model will be a useful drug-screening tool for cataract prevention and treatment (Marmor et al. 1990). The CRISPR/Cas9 system has been widely applied in genetically modified organisms, such as transgenic zebrafish (Chang et al. 2013), mouse (Zhou et al. 2014), rat (Ma et al. 2014) and human cells (Jinek et al. 2013).

CRISPR/Cas9-mediated gene therapy

Retinitis pigmentosa (RP)

RP is a severe retinal dysfunction disease that currently lacks an approved effective treatment. Gene therapy, as a powerful strategy to treat monogenetic retinal diseases, has rapidly developed in recent years. At the first stage, gene therapy was used to correct the loss of function and restore a normal phenotype through gene augmentation by delivery of a normal copy of the gene. Several studies have been carried out to design and develop a gene therapy vector for X-linked RP (XLRP) caused by mutations in RPGR (Beltran et al. 2017; Fischer et al. 2017; Pawlyk et al. 2016). Codon optimization has been successfully used as approach to produce RPGR gene therapy vectors without generating new mutations. Promising preclinical tests using AAV8.GRK1.coRPGR led to the initiation of a phase I/II clinical trial in subjects with XLRP caused by mutations in RPGRORF15 (Camara et al. 2018). As CRISPR/Cas9-mediated gene therapy can introduce or knock out multiple genes simultaneously, it is more powerful for multiplex gene editing. Latella et al. successfully edited the human RHODOPSIN (RHO) gene in a mouse model of autosomal dominant RP by CRISPR/Cas9 technology, confirming its efficacy as a genetic engineering tool in photoreceptor cells (Latella et al. 2016). Moreover, another study showed that the CRISPR/Cas9 system can be used in vivo to selectively ablate the Rhodopsin gene carrying the dominant S334ter mutation (Rho S334) in rat models with severe autosomal dominant RP. The allele-specific disruption of Rho S334 was generated by electroporation in the rat model, following a single subretinal injection of gRNA/Cas9 plasmid that prevented retinal degeneration and improved visual function (Benjamin et al. 2016).

Leber congenital amaurosis (LCA)

Bennicelli et al. used an AAV vector to deliver the RPE65 transgene in animal models of the RPE65 form of LCA (LCA2) and observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice (Bennicelli et al. 2008). In another study, AAV-mediated RPE65 gene transfer by subretinal injection was also used to treat dogs affected with disease caused by a Rpe65 deficiency. After long-term observation, stable restoration of rod and cone photoreceptor function in these dogs was observed, which has important implications for the treatment of human patients affected with LCA caused by RPE65 mutations (Acland et al. 2001, 2005).

Age-related macular degeneration (AMD)

AMD is the leading cause of irreversible blindness in people 50 years of age or older. For wet-AMD, choroidal neovascularization (CNV) is a major feature caused primarily by angiogenic cytokines, such as vascular endothelial growth factor A (VEGF-A), and results in further damage of the retinal structure and function (Jager et al. 2008). Kim et al. decreased the expression of Vegf-A and hypoxia inducible factor-1 alpha (Hif-1a) in vivo through Cas9 ribonucleoprotein (RNP)-mediated genome editing in an AMD mouse model. The area of laser-induced CNV in the mouse model of AMD was reduced. Their results showed that genome surgery through inactivating a disease-causing wild-type gene using Cas9 RNPs has local target treatment potential for nongenetic degenerative diseases such as AMD (Kim et al. 2017).

CRISPR/Cas9 genome editing combined with iPSCs

Stem cells are ideal for retinal cell replacement because they have the ability to self-renew and the potential to differentiate into multiple cell types. Currently, the main sources of replacement cells are retinal cells derived from retinal stem cells (RSCs), embryonic stem cells (ESCs), induced pluripotentstem cells (iPSCs), etc. (Tucker et al. 2014). In contrast to ESCs, iPSCs are autogenously derived to avoid immune rejection problems, enabling them to have broader application possibilities. Tucker et al. transplanted photoreceptor cells derived from adult mouse iPSCs into degenerative hosts, and they observed that the transplanted cells integrated into the retinal outer nuclear layer of the mice with retinal degeneration and developed into mature photoreceptor cells, giving rise to increased retinal function. The results above demonstrated that adult fibroblast-derived iPSCs can differentiate into retinal precursors to be used for transplantation and treatment of retinal degeneration diseases (Tucker et al. 2011). Similarly, Homma et al. also concluded that iPSC-derived rods in vitro may provide a renewable cell source for cell replacement therapy (Homma et al. 2014). Since patient-derived iPSCs carry pathogenic genes that may affect the survival and function of transplanted cells, it is necessary to edit disease-causing mutations before transplantation. As such, CRISPR/Cas9 technology can serve as a tool to be combined with iPSCs for basic research and clinical applications in gene therapy. Zheng et al. suggested that the combination of iPSCs with CRISPR gene editing could provide personalized therapy in which a patient’s own cells are corrected and used to replace their degenerating retina (Zheng et al. 2015). Bassuk et al. used the CRISPR/Cas9 system to precisely repair an RPGR point mutation that causes X-linked RP (XLRP) in patient-specific iPSCs, supporting the development of personalized iPSC-based transplantation therapies for retinal disease. Their study combined CRISPR/Cas9-mediated gene editing with autologous iPSCs to develop a personalized transplantation strategy that could be applied to various retinal diseases (Bassuk et al. 2016).

In addition, because they are derived from patients, iPSCs are highly suitable for disease modeling and disease pathophysiological investigations. Cereso et al. used AAV2/5 as a carrying vector to effectively transduce iPSC-derived RPE cells from a choroideremia patient, thus demonstrating the superiority of AAV2/5 in human RPE cells and illustrating the potential of patient iPSC-derived RPE cells to provide a proof-of-concept model for gene replacement when there is no appropriate animal model (Cereso et al. 2014). Mutations in CEP290 are the most common cause of LCA. Burnight et al. transduced patient-specific, iPSC-derived, photoreceptor precursor cells with lentiviral vectors carrying full-length CEP290 to correct the CEP290 disease-specific phenotype. Their results showed the expression of full-length transcripts and functional rescue of the ciliogenesis defect in patient cells, which is an LCA cellular CEP290 disease-specific phenotype (Burnight et al. 2014).

Progression and clinical transformation

CRISPR/Cas9target gene activation (TGA) system: turning genes on instead of snipping them

DSBs will be induced in the genome area that is targeted for editing or deletion in most CRISPR/Cas9 systems, potentially increasing the risk of introducing undesired mutations with deleterious effects. For this problem, Liao et al. reported a robust system that can transcriptionally activate target genes in vivo by modulating histone marks rather than editing DNA sequences to generate physiologically relevant phenotypes. The in vivo TGA system can activate target genes through trans-epigenetic remodeling, relying on the recruitment of Cas9 and transcriptional activation complexes to target loci by modified sgRNAs, known as the CRISPR/Cas9 TGA system or CRISPRa. In their study, CRISPR/Cas9-mediated TGA was achieved in mouse models of diabetes, muscular dystrophy, and acute kidney disease, leading to measurable phenotype improvements and disease symptom amelioration (Fig. 3). This new system affects gene activity via “epigenetics” instead of changing the DNA sequence, paving the way for developing targeted epigenetic therapies to treat human diseases (Liao et al. 2017). The new activator system will be useful for research, but some challenges remain to be solved before the therapy can be used in the clinic. The technology’s safety and efficacy must also be demonstrated, such as whether host immune responses against the AAV-CRISPR/Cas9 TGA system arise. Moreover, no paper has reported the application of CRISPRa in ophthalmological diseases, although such applications represent a new direction for CRISPR genome-editing technology development, which holds great promise as a targeted epigenetic approach for treating human diseases.

Fig. 3

In vivo delivery of a Cas9-based epigenetic gene activation system ameliorates disease phenotypes in mouse models of type I diabetes, acute kidney injury, and muscular dystrophy

(reprinted from Liao et al. (2017), Copyright (2017), with permission from Elsevier)

Clinical transformation

Editas Medicine conducted a Phase 1 clinical trial for LCA type 10 in 2017. They knocked out the disease-causing mutation and restored function with the CRISPR/Cas9 system, delivered via a local injection of AAV. This is the first time that CRISPR technology has been used in a clinical research project. In 2018, a US company named “CRISPR Treatment” became the first licensed organization from European regulators to conduct clinical trials. The company plans to solve genetic defects in beta thalassaemia patients using autologous hematopoietic stem cell therapy edited by CRISPR technology. The company also plans to apply for approval from the US Food and Drug Administration (FDA) to conduct a clinical trial to test CRISPR treatment for sickle cell disease next year.


Collectively, current studies show that the CRISPR/Cas9 system could be a useful and powerful tool for genome editing in targeting retinal degeneration diseases, not only in generating animal disease models but also in gene therapy in vivo. Moreover, the combination of CRISPR/Cas9 technology with iPSCs has promising applications in gene and cell therapy for retinal degeneration diseases; further investigations are needed in the future. Nonetheless, as a new technology that is rapidly advancing, applications in treating human genetic diseases are still confronted with many difficulties and challenges. It is imperative to develop methods to detect and decrease off-target effects and to further improve the efficiency of genome editing in vivo before CRISPR/Cas9 can be used clinically.



We thank Jin Yang, Xiaomin Zhang and Xiaorong Li for comments and suggestions, Jin Yang and Zhiqing Li for editing the manuscript, Bincui Cai for writing the manuscript, and Shuo Sun for assistance in generating the table. This work was supported by the National Natural Science Foundation (81670875; 81500745), China; the Natural Science Foundation of Tianjin City (18JCQNJC10700); the Natural Science Foundation of Tianjin City (17JCYBJC27200); and a grant from the Dr. Henry Norman Bethune: LangMu Young Scientist Scholarship (BJ-LM2015008L).


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Tianjin Medical University Eye HospitalTianjinChina

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