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CRISPR/Cas9 Genome-Editing System in Human Stem Cells: Current Status and Future Prospects

Genome-editing involves the insertion, deletion, or replacement of DNA in the genome of a living organism using “molecular scissors.” Traditional genome editing with engineered nucleases for human stem cells is limited by its low efficiency, high cost, and poor specificity. The CRISPR system has recently emerged as a powerful gene manipulation technique with advantages of high editing efficiency and low cost. Although this technique offers huge potential for gene manipulation in various organisms ranging from prokaryotes to higher mammals, there remain many challenges in human stem cell research. In this review, we highlight the basic biology and application of the CRISPR/Cas9 system in current human stem cell research, discuss its advantages and challenges, and debate the future prospects for human stem cells in regenerative medicine.

Genome-editing involves the insertion, deletion, or replacement of DNA in the genome of a living organism using "molecular scissors." Traditional genome editing with engineered nucleases for human stem cells is limited by its low efficiency, high cost, and poor specificity. The CRISPR system has recently emerged as a powerful gene manipulation technique with advantages of high editing efficiency and low cost. Although this technique offers huge potential for gene manipulation in various organisms ranging from prokaryotes to higher mammals, there remain many challenges in human stem cell research. In this review, we highlight the basic biology and application of the CRISPR/Cas9 system in current human stem cell research, discuss its advantages and challenges, and debate the future prospects for human stem cells in regenerative medicine.
Background CRISPR is a short and repeating nucleotide initially found in the genome of bacteria and archaea, and it functions to eliminate exogenous genetic elements (EGEs) that combine with Cas proteins. 1,2 Three steps are required to eliminate EGEs: new spacer integration, CRISPR/Cas combination, and degeneration ( Figure 1). First, some short nucleotides of invaders may integrate with the CRISPR loci of the host as new spacers. The CRISPR RNAs (crRNAs) are then transcripted to generate a crRNA/Cas complex. Finally, the EGEs will be inactivated by the complexes under the base complementation pairing rule. To date, three types of CRISPR-Cas immune system have been identified with different CRISPR repeat sequences and Cas protein profile. [3][4][5] Among them, the type II CRISPR-Cas9 immune system offers strong potential in developing a totally novel genome-editing tool for biological and medical study because it utilizes RNase III in crRNA transcription and requires just one Cas9 protein to form a crRNA/Cas9 complex. 5 Almendros's group 6 identified the conservative NGG motif, proto-spacer adjacent motif (PAM), after comparing the CRISPR array of Streptococcus pyogenes. In 2013, two articles revealed a new genome-editing stage for biological and biomedical studies. 7,8 Principle Guide RNA (gRNA) and Cas9 nuclease are core components of the CRISPR/Cas9 genome-editing system ( Figure 2). Unlike the original type II CRISPR-Cas immune system, its crRNA is eventually simpli-fied to the shortest mature and functional structure without the original crRNA processing. 7,8 This simplified gRNA contains two parts: a variable region and a basic scaffold. The former normally is composed of 18-20 nucleotides that can bind the target DNA according to the base complementation pairing rule. The latter is a long scaffold-like RNA used to bind Cas9 nuclease and form a gRNA/Cas9 complex. Then, the genome-editing system still requires three nucleotides containing a PAM, NGG. 6 Only the genome DNA that contains PAMs can be identified and bound by the gRNA/Cas9 complex to generate double-strand breaks (DSBs).

Advantages
The CRIPSR/Cas9 genome-editing system offers several advantages over the zinc-finger nucleases (ZFNs) and transcription activatorlike effector nuclease (TALEN) in human pluripotent stem cells (PSCs) and somatic stem cells (SSCs). First, CRISPR/Cas9 is more user-friendly than ZNF and TALEN. Several gRNAs such as synthesizing primers need to be selected for using CRISPR/Cas9, because its specificity is related only to ribonucleotide complex formation. 7,8 Second, CRISPR/Cas9 is more economical because there is little associated cost for plasmid-mediated CRISPR/Cas9. Third, as the fastest currently available genome-editing technique, genome editing using CRISPR/Cas9 can typically be achieved in 2 weeks. 9 Finally, CRISPR/Cas9 shows a higher editing efficiency than TALEN in human stem cells. Ding et al. 10 demonstrated the highest genome-editing efficiency of 79% using CRISPR/Cas9 to edit human PSCs. stem cells have been approved for clinical treatments, and many have demonstrated remarkable outcomes in clinical trials. 12 Increasing reports confirm that CRISPR/Cas9 genome editing is a powerful tool that can promote stem cell research, from basic biological to translational studies (Table 1).

Basic Biological Studies
Gene Knockout. The basic biology of PSCs has always been a fundamental component of stem cell and human developmental research. In addition, the high differentiation capacity of human PSCs enables their broad application. It is therefore essential to explore the intrinsic connection between the upstream regulatory mechanism and the downstream biological features. This can be achieved by adopting a classic gene knockout strategy. CRISPR/Cas9 can be used to rapidly induce gene mutations in human PSCs without changing their genetic background, making CRISPR/Cas9 a superior technique to other gene-interfering tools (ZFN, TALEN, and RNAi). 9 When a cell's genome DNA is broken by a gRNA/Cas9 complex, the genome repair system is activated. One such system is non-homologous end joining (NHEJ). This will directly ligate the broken DNA and result in the chance of introducing a wrong base-pair deletion or insertion for gene knockout. 9 Batista et al. 13 used CRISPR/Cas9 to knock out Mettl3 in order to erase m6A modification in human embryonic stem cells (ESCs), which prevented the self-renewal of ESCs and

New spacer genera on
Repeat Spacer Repeat Spacer Cas C C C C C C C C C C Cas as as as as as as as as as as as  19 fused a doxycycline-inducible dCas9 with KRAB and achieved conditional and reversible interference in human induced PSCs (iPSCs) and its derived somatic cells such as cardiac progenitors, cardiomyocytes, and T lymphocytes. Guo et al. 20 designed a doxycycline-inducible dCas9-VPR cassette to activate gene expression in human PSCs. These CRISPRi/a platforms should provide a more convenient strategy to explore gene functions and signaling pathways in human stem cell research, which are faster, more convenient, and more economical than other techniques such as RNAi and gene overexpression. 21 To achieve this, it is necessary to ligate primer-like gRNAs into plasmids to achieve rapid transcription activation or suppression without setting various controls or cDNA cloning, both of which are time consuming in RNAi or gene overexpression. For example, Liu et al. 22 utilized CRISPRi to interrupt the expression of long non-coding RNA (lncRNA) LOC646329 to reveal its key function in the proliferation of radial glial cells. Luo et al. 23 found that suppression of miR-199a/214 cluster could significantly increase the tumor tropism in human iPSC-derived neural stem cells (NSCs). Similarly, CRISPRa may improve survivability of stem cells by controlling their gene expression following transplantation. 24 CRISPRi/a is highly specific in targeting human stem cells. But because it targets transcription, it cannot be utilized in the study of alternative splicing.
Genome-wide Screening. CRISPR/Cas9 has been used for genomewide and high-throughput genetic screening in mammalian cells. 25,26 gRNA libraries have been generated to provide large volumes of genes for analyzing results through sequencing data collection and offer an alternative to the traditional RNAi library. Nonetheless, there may be differences in the response of the gene regulatory network, because RNAi knocks down gene expression at the mRNA level, whereas CRISPR/Cas9 targets gene knockout or transcription inhibition. 27 Technically, a virus mixture that contains thousands of gRNAs is produced to co-infect human stem cells with Cas9-or dCas9-expressed virus to simultaneously target thousands of genes to screen for those of interest. CRISPR/Cas9-mediated genome-wide screening strongly supports the basic biological research of human stem cells. Shalem et al. 28 built a CRISPR/Cas9 lentiviral library to simultaneously knock out 18,080 genes in human ESCs and identified many essential genes that are involved in cell survival. Liu et al. 29 identified 326 functional loci of lncRNAs in human iPSCs using the CRISPRi lentivirus library. It is clear that this genetic screening platform has the potential to reveal novel functional genes and a particular signaling pathway, 30 and should be a powerful platform for stem cell research.
Gene Knockin. Specific markers are extremely important for stem cell research and indispensable during stem cell differentiation, transplantation in vivo, and in vitro tracing. Normally, identification of a specific marker requires substantial time and energy, but CRISPR/ Cas9 has enabled vast improvements. It is now possible to easily and rapidly create a specific marker by fusing epitope tags or fluorescent proteins to the genes of interest. 31,32 With the exception of NHEJ, homology-directed repair (HDR) is another mechanism by which exogenous nucleotide sequences can be introduced into the genome through homologous recombination. 33 DSBs caused by CRISPR/ Cas9 significantly promote the homologous recombination between a targeted genome and exogenous DNA fragments to activate HDR  . Schemata of CRISPR/Cas9 Genome-Editing System gRNA contains a variable region (red nucleotides) and a basic scaffold structure (blue nucleotides). The former is complementary with one strand of targeted genome DNA that should be adjacent to PAM (NGG, green one). The basic scaffold nucleotide is then targeted to combine and support Cas9 nuclease to generate gRNA/Cas9 complexes. Finally, the targeted genome DNA will be cut at the third nucleotide from PAM (between G and C).
at a cell level. 9 Other Applications. Genomic imaging is indispensable to genetic and cell biology study. CRISPR/Cas9 can also be used to visually monitor the status of the genome during the cell cycle. Chen et al. 37 modified the original nucleotide sequences of gRNA scaffold to improve its binding ability with genome DNA, and fused the GFP to the C terminus of dCas9 nuclease to finally form a live image to monitor the dynamics of the genome in the cell cycle. This technique can image fixed or living cells with single or multiple colors. It may also be used to monitor the genomic activity of stem cells. Anton et al. 38 imaged the chromatin of mouse ESCs using this method.

Disease Model and Drug Screening
Traditional animal disease models are important to human biological and biomedical studies. Nonetheless, it is difficult to simulate the real situation in a human body because of species differences. Human PSCs can overcome this limitation. CRISPR/Cas9 helps to easily and rapidly introduce a gene mutation into human stem cells to model a human disease, and avoids ethical problems and those associated with species differences. Indeed, the CRISPR/cas9 system has been shown to be an effective method for drug screening in human stem cell disease models (Table 1).
Human PSCs. Human PSCs, including ESCs and iPSCs, are ideal targets for gene knockout because they are easily expanded for further experiments from a single cell culture after resistance selection without losing their pluripotency. 9 One straightforward strategy is to knock out disease-relevant genes in human PSCs using CRISPR/ Cas9 in order to explore the pathogenic mechanism in the derived cells (somatic cells, SSCs, and even tumor cells) and for drug screening. Rb1 is known to initiate retinoblastoma in humans. 39 RB1 null human ESCs generated by CRISPR/Cas9 showed aberrant mitochondria and were shown by drug screening to be sensitive to carboplatin. 40 Moreover, the isogenic human PSC knockout disease model is crucial to eliminate the individual variations and unknown gene mutations that occur during iPSC reprogramming. 41 Chang et al. 42 demonstrated different drug responses among cells derived from different patient-specific iPSCs and CRISPR/Cas9-generated isogenic PSCs. Employing CRISPR/Cas9 can precisely introduce mutations (point mutation, insertion, or deletion) into human PSCs to generate gene mutant PSCs and provide a patient disease model. It bypasses the limitations of iPSC generation that requires biopsy or reprogramming, and provides a strategy to determine precisely why different patients with the same mutations present with different features during pathogenesis. Guo et al. 43 used CRISPR/Cas9 to induce a point mutation (c.1288G>T) in the Men1 gene of normal iPSCs to study the effect of individual differences in hypoglycemia.   Gene Correction Therapy. Many gene mutations at the genome level often result in genetic disorders. Although allografts have been widely utilized in preclinical experiments and clinical cell therapy studies, immune rejection remains a concern. 55,56 CRISPR/Cas9-mediated gene correction provides a new vision of cell therapy for many human genetic diseases. The general strategy is to break the genomic DNA near the targeted genes using CRISPR/Cas9 and simultaneously provide a donor that presents normal sequences. The mutant gene fragments can be corrected through homologous recombination, and the associated functional defect in the targeted cells is corrected. It has been demonstrated in an animal model that gene correction offers a definitive cure for genetic disease. Li's group 57 co-injected gRNA, Cas9 nuclease, and healthy donor plasmid of the Crygc gene into the zygotes of cataract mice and generated cataract-free mice. Patient-specific iPSCs are the most common source for gene correction because they can be differentiated into all kinds of functional somatic cells after gene correction. Gene-corrected b-thalassemia patient-specific iPSCs are able to restore HBB expression and be differentiated into functional hematopoietic progenitor cells. 58,59 Patient-specific organoids offer a promising source for gene correction and are more easily differentiated into somatic cells for transplantation compared with PSPs. Schwank et al. 50 successfully obtained function recovered cells after they repaired the mutant CFTR gene in human intestinal stem cell organoids. For therapeutic purposes, stem cells should be maintained in a "stemness" state. Hence we normally do not criticize the purity of manipulated cells, and use adeno-associated virus (AAV) to deliver the donor for gene correction. Porteus's group 60,61 finally obtained around 38% biallelic-corrected b-thalassanemia HSCs using AAV-assisted gene correction after rough purification. This was sufficient for cell transplantation and avoided the difficulties caused by random integration in lentivirus or retrovirusmeditated gene therapy. 60,61 CRISPR/Cas9-mediated gene correction has become a principal therapeutic strategy in the study of genetic diseases such as b-thalassemia, Duchenne muscular dystrophy, hemophilia A, Huntington's disease, and X-linked chronic granulomatous disease. 58,[62][63][64][65] It should nonetheless be noted that although we can obtain functionally recovered stem cells after gene correction in vitro, their therapeutic safety and efficacy after transplantation remain to be evaluated.
Anti-virus Therapy. Some viruses bind to the surface receptors of host cells during infection. As mentioned above, CRISPR/Cas9 can help modify the targeted cells efficiently and easily. Hence infection with HIV can be prevented by modifying the receptors of human cells. For example, CCR5 and CXCR4 are co-receptors of human white blood cells for HIV infection. 66,67 There are reports showing that CCR5-or CXCR4-disrupted human CD4 + T cells, HSCs or iPSCs, present HIV resistance after CRISPR/Cas9-mediated gene modification. [68][69][70][71] Nevertheless, cells acquired HIV resistance through direct gene disruption, and some basic functions may also have been affected. It may be advisable to first define some natural mutations that do not affect the host's biological functions before utilizing CRISPR/Cas9 to introduce desirable mutations (e.g., point mutation) in cell engineering. Ye et al. 72 introduced a natural mutation (CCR5D32) into normal human iPSCs and finally obtained HIV-resistant monocytes and macrophages after cell differentiation.
In addition, CRISPR/Cas9 has been demonstrated to be effective in targeting the genetic material of viruses. 73 Liao et al. 74

engineered human iPSCs to express HIV-targeted gRNA and Cas9 protein that could protect cells from virus infection by disrupting the RNA of HIV in iPSCs and their derived adult cells.
Anti-tumor Therapy. Genetically engineered human immune cells and stem cells have already been widely used in anti-tumor research, such as delivering anti-tumor proteins and inducing cancer cell death. 75 Chimeric antigen receptor (CAR) T cells are considered a milestone in the cure of refractory blood cancer. 76 CRISPR/Cas9 can be used to increase their anti-tumor efficacy by disrupting the programmed death protein 1 (PD-1) in CAR T cells by eliminating PD-L1 (PD-1 ligand) expressed tumor xenografts in vivo. 77 In addition, CRISPR/Cas9 can be utilized to eliminate the risks associated with lentivirus-mediated CAR T cells, such as the potential of oncogenesis and recovery of replication. Eyquem et al. 78 engineered T cells to express CAR uniformly by directly fusing CD19 CAR to cells' TRAC gene and also increased their potency. For the treatment of solid tumors, Blum et al. 79 introduced the stable expression of CD16a (S197P) and disrupted ADAM17 in human iPSCs that could further differentiate into natural killer cells for anti-tumor therapeutic research.

Off-Target
A common problem of all gene manipulation techniques, including CRISPR/Cas9 systems, is that they miss their target. The mismatch between gRNA and genomic DNA is the main cause of non-specificity in genome editing. It has been shown that Cas9 nuclease has diverse tolerance to the mismatch between gRNA and targeted genomic DNA. The PAM mismatch (e.g., RNG) can also lead to being off-target. 80,81 The ratio of being off-target is relative to the targeted genes and hosts. For example, CRISPR/Cas9 can generate off-targets at a different ratio for different genes (60% for HBB gene and 22% for CCR5 gene) in the HEK293 cell line, while the ratio of off-targets is just 0.34% for the RNF2 gene in the HeLa cell line. 82,83 Off-targets can also be identified in stem cells. Wu et al. 84 revealed that off-target sites were mainly related to genes in mouse ESCs. Fortunately, recent whole-genome sequencing results have indicated that the incidence of off-targets caused by CRISPR/Cas9 is very low in human stem cells. [85][86][87] Although some inaccuracy is inevitable, it can be controlled or reduced using several common strategies.
Optimize gRNA Design. Well-designed gRNAs play very critical roles in ensuring a high efficiency of genome cleavage and low incidence of off-targets in human stem cell research. There are various online tools for gRNA design that are user-friendly. [88][89][90][91][92][93][94] When sequences of targeted genes are uploaded, all gRNAs that contain PAM (NGG) will be generated automatically, and the possibility of being off-targets is evaluated. Under normal circumstances, a higher score means a lower possibility. Therefore, the gRNAs with high scores are recommended in order to reduce the number of off-targets. Nevertheless, deep sequencing or nuclease detection is required to examine the incidence of off-targets in stem cells. Some studies have shown that shorter gRNAs can reduce the occurrence of offtarget by reducing their mispairing with the targeted genome, but the indels of on-target will also decrease accordingly, because binding of shorter gRNAs to the targeted genome is not as stable as that of longer ones. 82,83 Modify Cas9 Nuclease or gRNA. Cas9 nickase (Cas9n) is a mutant version of Cas9 that generates just a nick on the gRNA-bound strand without breaking the whole double-strand DNA. 95 This system needs a pair of gRNAs to form two gRNA/Cas9n complexes for DNA cleavage and can dramatically decrease the off-target in human ESCs. 96 Kleinstiver et al. 97,98 modified the traditional Streptococcus pyogenes Cas9 (SpCas9) to identify other PAM sequences or improve fidelity. Fu et al. 99 reduced off-targets 5,000-fold by using truncated gRNAs.
Exploit Alternatives to Classic SpCas9. Type I and type III CRISPR-Cas immune systems share a similar mechanism to type II. Tsai et al. 100 utilized a dimeric RNA to guide the highly specific cleavage of FokI nucleases based on the classic CRISPR/Cas9 system. Cas3 and Cmr are nucleases of the type I and type III CRISPR-Cas immune system and show better specificity than Cas9. 101,102 More encouragingly, no off-target has been detected using the CRISPR/ Cpf1 system. 103,104 Editing Efficiency Editing efficiency is another important aspect of gene manipulation techniques. CRISPR/Cas9 has been widely utilized and has shown high genome-editing efficiency in various mammalian cells. 105 In addition, the high editing efficiency can significantly reduce costs when processing stem cells in vitro. Nonetheless, its genome-editing efficiency is insufficient for human stem cell research and hampers the transfer of genome editing from the bench to the bedside. For example, a virus is commonly used for CRISPR/Cas9 delivery, but this confers risks for the clinical transplantation of stem cells. It is almost impossible for some stem cells, such as HSCs and MSCs, to be expanded sufficiently for clinical application from screening a single cell after the manipulation of CRISPR/Cas9.
Optimize the Expression of gRNA/Cas9. Cas9-induced genome editing is related to gRNA expression and gRNA/Cas9 binding. 106 Employing purified gRNA and Cas9 protein has been shown to directly improve the genome-editing efficiency in human stem cells and provide more stable and sufficient Cas9 nuclease for genome editing. Kim et al. 107 delivered purified gRNA and Cas9 into human H9 ESCs, and finally achieved 23% indels, which was almost five times higher than that reported by Mali et al. 8 in 2013. Gundry et al. 108 remarkably enhanced the genome-editing efficiency of CRISP/Cas9 in human HSCs using purified gRNA and Cas9 nuclease. This avoids the effect of protein transcription and translation. In addition, editing efficiency can be improved by controlling the delivery time of gRNA and Cas9 nuclease in 293 cells, and may also be appropriate for stem cells. 109 Modify gRNAs or Cas9 Nucleases Artificially. Longer gRNA binds to Cas9 nuclease more tightly with a consequent huge improvement in genome-editing efficiency. 105 Exogenous RNAs cannot stay long in human cells because of RNA degeneration. 110 Genome-editing efficiency can be improved if gRNA can be maintained for a longer time prior to degradation. Hendel et al. 111 utilized some chemical groups to protect the phosphoramidites of gRNAs and significantly enhanced the genome-editing efficiency of CRISPR/Cas9 in T cells and CD34 + HSCs. Other artificially modified Cas9 nuclease or alternatives to CRISPR/Cas9 have also been reported to have greater genome-editing efficiency, such as Cas3-CRISPR, type III-A CRISPR-Cas-Csm, FokI-CRISPR, and Cpf1-CRISPR. [100][101][102]104 Nonetheless, stronger experimental evidence is required to support these alternatives in stem cell studies.
Preprocess Host Cells. CRISPR/Cas9-mediated genome editing depends on NHEJ and HDR. Yu et al. 112 identified two compounds (L755507 and brefeldin A) that improved genome-editing efficiency by enhancing HDR efficiency in human PSCs. SCR7 is a small molecular inhibitor of the NHEJ pathway. 113 Zygotes treated with SCR7 showed a 19-fold improvement in gene knockin efficiency using CRISPR/Cas9. 114 Similarly, RS-1, an HDR enhancer, improved the gene knockin efficiency 2-to 5-fold in mouse embryos. 115 Other bioactive proteins, such as E1B55K and E4orf6, can likewise increase the knockin efficiency. 116 Although the toxicity of these small molecules and proteins requires extensive evaluation in human stem cells, they provide invaluable information to improve the editing efficiency of CRISPR/Cas9 in stem cell research.
Utilize Auxiliary Elements. Microhomology-mediated end joining (MMEJ) is another DNA repairing system in cells. 117 On this basis, a precise integration into a target chromosome (PITCh) system has been designed to improve the integration efficiency of gene knockin using several gRNAs to cut genome and PITCh vectors. 118 It is easier during donor construction, because the PITCh vector needs only a short micro homologous arm (5-25 bp) that can be synthesized directly. In contrast, classical homologous arms normally comprise thousands of base pairs of DNA amplified by PCR. Nonetheless, more experimental evidence is required to support its use in human stem cells. PiggyBac is a transposon system that can achieve change in position and insertion and deletion of DNA fragments from a chromosome. 119 It has been reported to dramatically increase CRISPR/Cas9-mediated genome-editing efficiency in human PSCs from the normal level of 1%-5% without using an integrated screening cassette. 58,120 Nonetheless, the random transposable action of PiggyBac transposons needs to be evaluated.

Delivery Routes
The commercial CRISPR/Cas9 genome-editing system is generally presented as plasmids, RNAs, or proteins. Nonetheless, this system cannot be used for gene manipulation unless they can be delivered into the targeted hosts. Selection of an appropriate delivery method is thus vital for CRISPR/Cas9-mediated gene manipulations. Normally, they have delivery strategies similar to other genemanipulating tools such as RNAi, ZFN, or TALEN. Nonetheless, because of the unique features of human PSCs and SSCs in regenerative medicine, different delivery strategies need to be considered carefully.
Transient Expression Strategies. Gene manipulation can be easily achieved by transient transfections of gRNA/Cas9 nuclease-expressed plasmids or their purified synthetic products in common cells. 7,8,107,121 The situation becomes more complex in stem cell studies as stem cells are more fragile, sensitive, or resistant to the transient delivery methods than common somatic cells and immortalized cell lines. Hence appropriate delivery methods are required. In terms of efficiency of delivery, micro-injection achieves the highest up to 100% for direct injection of substrates into targeted cells one by one through glass micro-pipettes. 122 But this micro-injection cannot be applied for large-scale injections, and thus limits its application to immortalized cells (e.g., PSCs). 123 Both electrotransfection and nucleofection have achieved high transfection efficiency in various human stem cells. The former can treat more kinds of cells in principle, while the latter presents higher cell viability after transfection. 58,[124][125][126] Millions of cells can be handled in one reaction based on electroporation; nonetheless, much time and effort have been expended in purifying the real transfected cells using selection cassettes. 58,124 Lipidosome reagents are not as popular as the other three methods for human stem cell transfection, because their transfection efficiency is poor when targeting many human stem cell lines. In addition, many human stem cell lines are extremely sensitive to the cytotoxicity of transfection reagents and the mechanical damage that occurs during cell pretreatments. Some new lipidosome-like delivery methods are now available with CRISPR/Cas9. Yin et al. 127 exploited a new lipid nano-particle to deliver Cas9 mRNA for genome editing; Jiang et al. 128  AAV-Mediated Strategy. Theoretically, AAV supports all applications of the CRISPR/Cas9 gene manipulating system similar to lentivirus (e.g., genome editing, CRISPRi/a, CRISPR imaging, and so on) because it shares the advantages of lentivirus in stem cell research. 136,137 We can easily obtain extremely high titering AAV virus using helper plasmids during virus packaging with consequent higher infection efficiency than lentivirus. 138,139 More importantly, an AAV-delivered donor is genome integration-free. It permanently exists as episomal DNA in cells and largely promotes efficiency of CRISPR/Cas9-mediated gene correction in stem cell therapy. [140][141][142] For example, Tabebordbar et al. 143 repaired mutant Dmd genes by AAV-delivered CRISPR/Cas9 in muscle stem cells. Unfortunately, the insert size capacity of AAV was far smaller than lentivirus.
With the exception of SaCas9 (Staphylococcus aureus), smaller nucleases need to be exploited to enhance editing efficiency when choosing AAV as the delivery vehicle. 144 In addition, AAV-delivered gRNA and Cas9 nuclease will be permanently expressed in all new divided cells. This may induce undesired genome damage and ultimately lead to cell death. 145 Although this situation has been resolved by delivering donor (AAV) and CRISPR/Cas9 (transient delivery strategies) separately, the advantages of AAV are partly concealed. 61 In addition, the immunogenicity of AAV remains another risk that must be considered. 146 Novel Delivery Strategies. With the development of CRISPR/Cas9, some novel delivery methods have emerged that eliminate the toxic effects or safety problems associated with transient transfection or virus infections. A chip containing micro-constrictions can generate transient membrane holes on cells by mechanical deformation to deliver CRISPR/Cas9 plasmids when the mixture of cells and plasmid pass the chip rapidly. 147 This method is reported to introduce high efficiency in genome editing with low cell loss in hard-to-transfect cells. 147 Based on the membrane punching mechanism, it also has the advantages and disadvantages of electroporation. The other methods are to utilize cell-penetrating peptides (CPP) to conjugate gRNA and Cas9 nuclease for CRISPR/Cas9 delivery in a plasmid-free, transfecting reagent-free, and virus-free manner. 121 A disadvantage is its relatively low transfection efficiency.

Prospects
Since its introduction in 2013, the CRISPR/Cas9 genome-editing system has been rapidly developed and widely used in all human stem cell studies. All gene-manipulating capacities (e.g., knockout, knockin, knockdown, and expression activating) are incredibly integrated in one technique. It is user-friendly, efficacious, and economical such that genome manipulation ceases to be a challenge for new researchers. Undoubtedly, the CRISPR/Cas9 genome-editing system has revolutionarily changed the fundamental and translational stem cell research. Although solutions are still required to reduce the off-target effect, improve editing efficiency, and exploit novel delivery strategies at a low cost and with high safety for clinical stem cell studies, CRISPR/Cas9-mediated preclinical studies have made remarkable progress and offer huge potential in human stem cell and regenerative research.

CONFLICTS OF INTEREST
The authors declare that they have no competing interests.