Induced Pluripotent Stem Cells Meet Genome Editing

“Lastly, demonstrate that the resulting induced pluripotent cells have all the key features of their embryonic stem cell counterparts, such as a stem celllike expression profile, the ability to give rise to differentiated cells in teratoma formation assays and their contribution to tissues in chimeric mice after blasto…”

“In 2012 the collaborative work of the Jennifer Doudna and Emmanuelle Charpentier laboratories demonstrated that in CRISPR type-2 systems a single protein, Cas9, can function as a designer SSN, by associating with an engineered single guide RNA (sgRNA) that bears homology to a genetic locus of interest (Jinek et al., 2…”

“Around the same time as the first mammalian SCNT efforts, James Thomson derived the first human embryonic stem cell lines (Thomson et al., 1998).”

“…and Cas9-mediated genome engineering was developed into the platform of choice to generate SSNs and to genetically modify hPSCs (Chen et al., 2015; Cong et al., 2013; Fu et al., 2014; Gonzalez et al., 2014; Gu et al., 2015; Hou et al., 2013; Hsu et al., 2014; Kleinstiver et al., 2016; Liao and…”

“…equally potent in reprogramming cells to a pluripotent state, providing valuable insights into the transcriptional pluripotency networks and how cells establish pluripotency (Buganim et al., 2012) (Apostolou and Hochedlinger, 2013; Park et al., 2008; Takahashi and Yamanaka, 2015; Yu et al., 2007).”

“…(Chen et al., 2015; Cong et al., 2013; Fu et al., 2014; Gonzalez et al., 2014; Gu et al., 2015; Hou et al., 2013; Hsu et al., 2014; Kleinstiver et al., 2016; Liao and Karnik, 2015; Lin et al., 2014; Mali et al., 2013; Ran et al., 2015; Slaymaker et al., 2016; Tsai et al., 2014; Wu et al., 2014b).”

“He used a very similar strategy that had proven successful for Evans and Martin (Evans and Kaufman, 1981; Martin, 1981), culturing the inner cell mass outgrowth of explanted blastocysts.”

“Organoid cultures are small functional tissue units comprised of several distinct cell types that can be maintained and used to recapitulate features of tissues rather than that of individual cell types in vitro (Lancaster et al., 2013; Sato et al., 2011; Sato et al., 2009) (reviewed in (Lancaster and Knoblich, 2014)…”

“…loss-of-function screens, for example, to identify gene products that are required for drug resistance or to mediate viral cell death (Gilbert et al., 2014; Hart et al., 2015; Parnas et al., 2015; Shalem et al., 2014; Shi et al., 2015; Wang et al., 2015a; Wang et al., 2014a; Zhou et Cell Stem Cell.”

“The low frequency of spontaneous homology mediated gene targeting in hPSCs was dramatically increased through the development of site-specific nucleases (SSN) as a tool for their genetic engineering (reviewed in (Carroll, 2014; Hsu et al., 2014; Urnov et al., 2010)).”

“He used a very similar strategy that had proven successful for Evans and Martin (Evans and Kaufman, 1981; Martin, 1981), culturing the inner cell mass outgrowth of explanted blastocysts.”

“Organoid cultures are small functional tissue units comprised of several distinct cell types that can be maintained and used to recapitulate features of tissues rather than that of individual cell types in vitro (Lancaster et al., 2013; Sato et al., 2011; Sato et al., 2009) (reviewed in (Lancaster and Knoblich, 2014)…”

“…loss-of-function screens, for example, to identify gene products that are required for drug resistance or to mediate viral cell death (Gilbert et al., 2014; Hart et al., 2015; Parnas et al., 2015; Shalem et al., 2014; Shi et al., 2015; Wang et al., 2015a; Wang et al., 2014a; Zhou et Cell Stem Cell.”

“…ease and scope of Cas9-mediated genome engineering in hPSCs was the establishment of CRISPR/CAS-systems from different organisms (Hou et al., 2013; Zetsche et al., 2015) that respond to different PAM sequences or by engineering spCas9 to associate with alternative PAMs by structure-based…”

“Most experiments that have detected significant off-targets have been performed in cancer cells, which may have altered repair pathways that could affect recombination (Fu et al, 2013; Hsu et al, 2013).”

“For the mouse system it was essential to establish that iPSCs could pass the most stringent test for pluripotency: germ line transmission (Maherali et al., 2007; Okita et al., 2007; Wernig et al., 2007) and tetraploid complementation (Kang et al., 2009; Zhao et al., 2009).”

“Organoid cultures are small functional tissue units comprised of several distinct cell types that can be maintained and used to recapitulate features of tissues rather than that of individual cell types in vitro (Lancaster et al., 2013; Sato et al., 2011; Sato et al., 2009) (reviewed in (Lancaster and Knoblich, 2014)…”

“In contrast, experiments in whole organisms such as mice ((Wang et al., 2013), primates (Niu et al., 2014), Zebrafish (Auer et al., 2014) or C. elegans (Dickinson et al., 2013) reported off-target frequencies that were low or not detectable consistent with high specificity of the CRISPR/Cas9…”

“Most experiments that have detected significant off-targets have been performed in cancer cells, which may have altered repair pathways that could affect recombination (Fu et al, 2013; Hsu et al, 2013).”

“…equally potent in reprogramming cells to a pluripotent state, providing valuable insights into the transcriptional pluripotency networks and how cells establish pluripotency (Buganim et al., 2012) (Apostolou and Hochedlinger, 2013; Park et al., 2008; Takahashi and Yamanaka, 2015; Yu et al., 2007).”

“…generate animals carrying defined mutations in multiple species including fish, drosophila, mice and primates (Bassett et al., 2013; Chang et al., 2013; Gratz et al., 2013; Hwang et al., 2013a; Hwang et al., 2013b; Niu et al., 2014; Wang et al., 2013; Yang et al., 2013; Yu et al., 2013).”

“…units comprised of several distinct cell types that can be maintained and used to recapitulate features of tissues rather than that of individual cell types in vitro (Lancaster et al., 2013; Sato et al., 2011; Sato et al., 2009) (reviewed in (Lancaster and Knoblich, 2014) (Sato and Clevers, 2013).”

“Parallel differentiation of such isogenic sets of cells into disease-relevant cells and tissues can be used to directly assess the contribution of a mutation to cellular pathology (Chung et al., 2013; Ryan et al., 2013; Wang et al., 2014b; Yusa et al., 2011).”

“…have been successfully derived by fusions with functional protein that bind specific loci, or to activate or repress gene activity at the target site (Chen et al., 2013; Gilbert et al., 2014; Konermann et al., 2015; Mandegar et al., 2016; Tanenbaum et al., 2014) (CRISPRa and CRISPRi, Figure 2).”

“…employed in loss-of-function screens, for example, to identify gene products that are required for drug resistance or to mediate viral cell death (Gilbert et al., 2014; Hart et al., 2015; Parnas et al., 2015; Shalem et al., 2014; Shi et al., 2015; Wang et al., 2015a; Wang et al., 2014a; Zhou…”

“…(Chen et al., 2015; Cong et al., 2013; Fu et al., 2014; Gonzalez et al., 2014; Gu et al., 2015; Hou et al., 2013; Hsu et al., 2014; Kleinstiver et al., 2016; Liao and Karnik, 2015; Lin et al., 2014; Mali et al., 2013; Ran et al., 2015; Slaymaker et al., 2016; Tsai et al., 2014; Wu et al., 2014b).”

“For the mouse system it was essential to establish that iPSCs could pass the most stringent test for pluripotency: germ line transmission (Maherali et al., 2007; Okita et al., 2007; Wernig et al., 2007) and tetraploid complementation (Kang et al., 2009; Zhao et al., 2009).”

“…Woltjen et al., 2009), by avoiding integration of the reprogramming factors all together using non-integrating viruses (Fusaki et al., 2009), episomal vectors (Yu et al., 2009) or direct transfection of the reprogramming factors as either mRNA (Warren et al., 2010) or protein (Kim et al., 2009).”

“…(Chen et al., 2015; Cong et al., 2013; Fu et al., 2014; Gonzalez et al., 2014; Gu et al., 2015; Hou et al., 2013; Hsu et al., 2014; Kleinstiver et al., 2016; Liao and Karnik, 2015; Lin et al., 2014; Mali et al., 2013; Ran et al., 2015; Slaymaker et al., 2016; Tsai et al., 2014; Wu et al., 2014b).”

“It appears that cell-intrinsic features such as low homologous recombination and single-cell survival rates make conventional genome modification as described by Capecchi and Smithies for mESCs (Doetschman et al., 1987; Thomas and Capecchi, 1987) very inefficient in hPSCs.”

“A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript whether iPSCs retain to some extent an epigenetic memory (Kim et al., 2010; Polo et al., 2010).”

“The low frequency of spontaneous homology mediated gene targeting in hPSCs was dramatically increased through the development of site-specific nucleases (SSN) as a tool for their genetic engineering (reviewed in (Carroll, 2014; Hsu et al., 2014; Urnov et al., 2010)).”

“Moreover, the GUIDE-Seq. protocol was also used to engineer CRISPR-Cas9 nucleases with altered PAM Cell Stem Cell.”

“For one, it became evident that many protocols that were developed for the differentiation of hPSCs into functional tissue resulted in embryonic rather than adult human cell types (Bedada et al., 2015; Forster et al., 2014; Hrvatin et al., 2014; Spence et al., 2011; Takebe et al., 2013).”

“…the developmental stage that is captured in vitro from the outgrowth of the inner cell mass, where hPSCs cultured under standard conditions represent a later epiblast-like pluripotent state (Brons et al., 2007; Tesar et al., 2007; Theunissen et al., 2014) and (reviewed in (Nichols and Smith, 2009).”

“Both of these challenges have been overcome: the development of the Rho-kinase inhibitor Y-27632 to suppress anoikis during the disaggregation of hPSC colonies dramatically increased single-cell survival of hPSCs (Watanabe et al., 2007).”

“…the developmental stage that is captured in vitro from the outgrowth of the inner cell mass, where hPSCs cultured under standard conditions represent a later epiblast-like pluripotent state (Brons et al., 2007; Tesar et al., 2007; Theunissen et al., 2014) and (reviewed in (Nichols and Smith, 2009).”

“Based on these data sets Tsai et al proposed that shorter guide sequences that only have about 17nt homology to the target sequence would improve specificity (Fu et al., 2014).”

“…have been successfully derived by fusions with functional protein that bind specific loci, or to activate or repress gene activity at the target site (Chen et al., 2013; Gilbert et al., 2014; Konermann et al., 2015; Mandegar et al., 2016; Tanenbaum et al., 2014) (CRISPRa and CRISPRi, Figure 2).”

“For one, it became evident that many protocols that were developed for the differentiation of hPSCs into functional tissue resulted in embryonic rather than adult human cell types (Bedada et al., 2015; Forster et al., 2014; Hrvatin et al., 2014; Spence et al., 2011; Takebe et al., 2013).”

“Decades later, the cloning of the sheep “Dolly” also by somatic cell nuclear transfer (SCNT) demonstrated that Gurdon’s finding extended to mammals as well (Campbell et al., 1996).”

“…the developmental stage that is captured in vitro from the outgrowth of the inner cell mass, where hPSCs cultured under standard conditions represent a later epiblast-like pluripotent state (Brons et al., 2007; Tesar et al., 2007; Theunissen et al., 2014) and (reviewed in (Nichols and Smith, 2009).”

“For example, this approach has been used successfully to identify the molecular principles underlying the most frequent non-coding mutations associated with human cancer (Bojesen et al., 2013; Fredriksson et al.; Horn et al., 2013; Huang et al., 2013; Killela et al., 2013).”

“, 2009) or the piggyBack system (Kaji et al., 2009; Woltjen et al., 2009), by avoiding integration of the reprogramming factors all together using non-integrating viruses (Fusaki et al.”

“For example, this approach has been used successfully to identify the molecular principles underlying the most frequent non-coding mutations associated with human cancer (Bojesen et al., 2013; Fredriksson et al.; Horn et al., 2013; Huang et al., 2013; Killela et al., 2013).”

“For the mouse system it was essential to establish that iPSCs could pass the most stringent test for pluripotency: germ line transmission (Maherali et al., 2007; Okita et al., 2007; Wernig et al., 2007) and tetraploid complementation (Kang et al., 2009; Zhao et al., 2009).”

“demonstrated that engineered zinc finger nucleases (ZFN) can serve as a designer SSN to correct X-linked SCID disease-relevant mutations in patientspecific cells (Urnov et al., 2005).”

“A key limitation remains that human cells prefer to choose the imprecise NHEJ pathway to repair a DSB rather than using the more precise homologous DNA repair pathway using an exogenous repair template (Chapman et al., 2012).”

“In less than four years this prediction became reality and Cas9-mediated genome engineering was developed into the platform of choice to generate SSNs and to genetically modify hPSCs (Chen et al., 2015; Cong et al., 2013; Fu et al., 2014; Gonzalez et al., 2014; Gu et al., 2015; Hou et al., 2013; Hsu et al., 2014; Klei…”

“While these data argue for the high specificity of Cas9, data in cancer cells suggest that offtargets can be frequently detected (Frock et al., 2015; Fu et al., 2014; Tsai et al., 2015; Wang et al., 2015b).”

“Such factor-free iPSCs were generated by independent methods such as the excision of reprogramming factors using the Cre/LoxP (Soldner et al., 2009) or the piggyBack system (Kaji et al., 2009; Woltjen et al., 2009), by avoiding integration of the reprogramming factors all together using…”

“…generate animals carrying defined mutations in multiple species including fish, drosophila, mice and primates (Bassett et al., 2013; Chang et al., 2013; Gratz et al., 2013; Hwang et al., 2013a; Hwang et al., 2013b; Niu et al., 2014; Wang et al., 2013; Yang et al., 2013; Yu et al., 2013).”

“…loss-of-function screens, for example, to identify gene products that are required for drug resistance or to mediate viral cell death (Gilbert et al., 2014; Hart et al., 2015; Parnas et al., 2015; Shalem et al., 2014; Shi et al., 2015; Wang et al., 2015a; Wang et al., 2014a; Zhou et Cell Stem Cell.”

“…have been successfully derived by fusions with functional protein that bind specific loci, or to activate or repress gene activity at the target site (Chen et al., 2013; Gilbert et al., 2014; Konermann et al., 2015; Mandegar et al., 2016; Tanenbaum et al., 2014) (CRISPRa and CRISPRi, Figure 2).”

“Combining the cellular versatility of iPSC differentiation with the ease of CRISPR/Cas9-mediated genome editing proved to be a very powerful experimental approach and by now genome editing in hPSCs has become a standard tool in stem cell research and human disease modeling (Johnson and Hockemeyer, 2015; Matano et al.,…”

“, 2009), by avoiding integration of the reprogramming factors all together using non-integrating viruses (Fusaki et al., 2009), episomal vectors (Yu et al.”

“For example, this approach has been used successfully to identify the molecular principles underlying the most frequent non-coding mutations associated with human cancer (Bojesen et al., 2013; Fredriksson et al.; Horn et al., 2013; Huang et al., 2013; Killela et al., 2013).”

“The rampant progress that has been made over the last few years to improve genome editing technologies and to detect and reduce potential off-targets of SSNs has already lead to the first clinical trials for HIV and are trail blazing through the necessary regulatory hurdles (Tebas et al., 2014).”

“GWAS have identified genomic loci that may slightly increase the risk of developing a sporadic disease.”

“A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript whether iPSCs retain to some extent an epigenetic memory (Kim et al., 2010; Polo et al., 2010).”

“Approaches to shift the balance away from NHEJ and towards homology-mediated repair included NHEJ inhibition with small molecules or by controlling the timing of CRISPR/Cas9 delivery with respect to the cell cycle stage (Chu et al., 2015; Maruyama et al., 2015; Robert et al., 2015; Yu et al., 2015).”

“Alternative strategies for increasing editing efficiencies include methods to more efficiently detect and subclone cells that have undergone rare editing events (Miyaoka et al., 2014) as well as to enhance delivery methods for the nuclease and donor template (Lin et al., 2014).”

“…units comprised of several distinct cell types that can be maintained and used to recapitulate features of tissues rather than that of individual cell types in vitro (Lancaster et al., 2013; Sato et al., 2011; Sato et al., 2009) (reviewed in (Lancaster and Knoblich, 2014) (Sato and Clevers, 2013).”

“Thus, these cells provide an experimental system to study pathogenesis of the disease in an in vitro system and to possibly devise therapeutic strategies (Robinton and Daley, 2012).”

“Approaches to shift the balance away from NHEJ and towards homology-mediated repair included NHEJ inhibition with small molecules or by controlling the timing of CRISPR/Cas9 delivery with respect to the cell cycle stage (Chu et al., 2015; Maruyama et al., 2015; Robert et al., 2015; Yu et al., 2015).”

“Combining the cellular versatility of iPSC differentiation with the ease of CRISPR/Cas9-mediated genome editing proved to be a very powerful experimental approach and by now genome editing in hPSCs has become a standard tool in stem cell research and human disease modeling (Johnson and Hockemeyer, 2015; Matano et al.,…”

“While technical challenges currently limit the potential application of such modifications, two recent papers describe gene editing of the embryo’s genome following injection of gRNAs, CRSPR/Cas9 RNA and targeting oligos into human zygotes (Kang et al., 2016; Liang et al., 2015).”

“…the use of SSNs such as ZFNs and transcription activator like effector nucleases (TALENs) to engineer hPSCs (DeKelver et al., 2010; Hockemeyer and Jaenisch, 2010; Hockemeyer et al., 2009; Hockemeyer et al., 2011; Lombardo et al., 2007; Sexton et al., 2014; Soldner et al., 2011; Zou et al., 2009).”

“, 2013), primates (Niu et al., 2014), Zebrafish (Auer et al.”

“…generate animals carrying defined mutations in multiple species including fish, drosophila, mice and primates (Bassett et al., 2013; Chang et al., 2013; Gratz et al., 2013; Hwang et al., 2013a; Hwang et al., 2013b; Niu et al., 2014; Wang et al., 2013; Yang et al., 2013; Yu et al., 2013).”

“Elegant work that also takes advantage of the fact that genomic sequences between two SSN cuts can reinsert into back into the locus in an inverted manner was recently used demonstrated that CTCF sites interact with each other in an orientation dependent manner (Guo et al., 2015).”

“…(Chen et al., 2015; Cong et al., 2013; Fu et al., 2014; Gonzalez et al., 2014; Gu et al., 2015; Hou et al., 2013; Hsu et al., 2014; Kleinstiver et al., 2016; Liao and Karnik, 2015; Lin et al., 2014; Mali et al., 2013; Ran et al., 2015; Slaymaker et al., 2016; Tsai et al., 2014; Wu et al., 2014b).”

“…(Chen et al., 2015; Cong et al., 2013; Fu et al., 2014; Gonzalez et al., 2014; Gu et al., 2015; Hou et al., 2013; Hsu et al., 2014; Kleinstiver et al., 2016; Liao and Karnik, 2015; Lin et al., 2014; Mali et al., 2013; Ran et al., 2015; Slaymaker et al., 2016; Tsai et al., 2014; Wu et al., 2014b).”

“…experiments in whole organisms such as mice ((Wang et al., 2013), primates (Niu et al., 2014), Zebrafish (Auer et al., 2014) or C. elegans (Dickinson et al., 2013) reported off-target frequencies that were low or not detectable consistent with high specificity of the CRISPR/Cas9 mediated…”

“…zygote and was used to efficiently generate animals carrying defined mutations in multiple species including fish, drosophila, mice and primates (Bassett et al., 2013; Chang et al., 2013; Gratz et al., 2013; Hwang et al., 2013a; Hwang et al., 2013b; Niu et al., 2014; Wang et al., 2013; Yang et…”

“…the use of SSNs such as ZFNs and transcription activator like effector nucleases (TALENs) to engineer hPSCs (DeKelver et al., 2010; Hockemeyer and Jaenisch, 2010; Hockemeyer et al., 2009; Hockemeyer et al., 2011; Lombardo et al., 2007; Sexton et al., 2014; Soldner et al., 2011; Zou et al., 2009).”

“…SSNs was met and resolved, similarly to the need for an easy way to make iPSCs, through a single experiment: by repurposing the bacterial Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) adaptive immune systems (reviewed in (Marraffini, 2015) as a SSN.”

“…applies to hPSCs as well as other systems such as C. elegans (Morton et al., 2006; Wood et al., 2011) and Drosophila melanogaster (Beumer et al., 2008; Bibikova et al., 2003; Bibikova et al., 2002), which are similarly resilient to conventional gene-targeting strategies as hPSCs. Cell Stem Cell.”

“…to efficiently generate animals carrying defined mutations in multiple species including fish, drosophila, mice and primates (Bassett et al., 2013; Chang et al., 2013; Gratz et al., 2013; Hwang et al., 2013a; Hwang et al., 2013b; Niu et al., 2014; Wang et al., 2013; Yang et al., 2013; Yu et al.,…”

“…equally potent in reprogramming cells to a pluripotent state, providing valuable insights into the transcriptional pluripotency networks and how cells establish pluripotency (Buganim et al., 2012) (Apostolou and Hochedlinger, 2013; Park et al., 2008; Takahashi and Yamanaka, 2015; Yu et al., 2007).”

“For the mouse system it was essential to establish that iPSCs could pass the most stringent test for pluripotency: germ line transmission (Maherali et al., 2007; Okita et al., 2007; Wernig et al., 2007) and tetraploid complementation (Kang et al., 2009; Zhao et al., 2009).”

“One approach to increase the maturity of in vitro cell systems and to mimic cellular aging is to expose these cells to stressors that are associated with aging (Miller et al., 2013; Studer et al., 2015).”

“It appears that cell-intrinsic features such as low homologous recombination and single-cell survival rates make conventional genome modification as described by Capecchi and Smithies for mESCs (Doetschman et al., 1987; Thomas and Capecchi, 1987) very inefficient in hPSCs.”

“…that a double strand break (DSB) generated by a SSN at a defined genomic site can be repaired either by the endogenous homology-mediated repair machinery using an exogenous provided repair template or by the error-prone Non-homologous end joining (NHEJ)-DNA repair pathway (Rouet et al., 1994a, b).”

“…to generate SSNs and to genetically modify hPSCs (Chen et al., 2015; Cong et al., 2013; Fu et al., 2014; Gonzalez et al., 2014; Gu et al., 2015; Hou et al., 2013; Hsu et al., 2014; Kleinstiver et al., 2016; Liao and Karnik, 2015; Lin et al., 2014; Mali et al., 2013; Ran et al., 2015; Slaymaker…”

“While these data argue for the high specificity of Cas9, data in cancer cells suggest that offtargets can be frequently detected (Frock et al., 2015; Fu et al., 2014; Tsai et al., 2015; Wang et al., 2015b).”

“…mediated editing of genomes proved to be almost universal and applies to hPSCs as well as other systems such as C. elegans (Morton et al., 2006; Wood et al., 2011) and Drosophila melanogaster (Beumer et al., 2008; Bibikova et al., 2003; Bibikova et al., 2002), which are similarly resilient to…”

“A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript hPSCs (Zwaka and Thomson, 2003).”

“Independent proof of concept studies demonstrated that SSNs can be used to repair or introduce disease relevant mutations in hPSCs (Soldner et al., 2011; Yusa et al., 2011).”

“Excitingly more applications are being developed; recently Cas9 has now also been programmed to target RNA in vitro and in vivo (O’Connell et al., 2014) (Nelles et al.”

“In contrast, experiments in whole organisms such as mice ((Wang et al., 2013), primates (Niu et al., 2014), Zebrafish (Auer et al., 2014) or C. elegans (Dickinson et al., 2013) reported off-target frequencies that were low or not detectable consistent with high specificity of the CRISPR/Cas9…”

“They came at a time when the potential of pluripotent stem cells in research applications and regenerative medicine had widely been appreciated (Rideout et al., 2002) (Figure 1), but technical and ethical limitations presented a challenge that severely impeded major progress towards realizing their full potential.”

“A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript specificities (Kleinstiver et al., 2015a; Kleinstiver et al., 2015b) and reduced off-targets (Kleinstiver et al., 2016).”

“Indeed, the number of human diseases modeled in culture using patient derived iPS cells (“disease in the dish”) is growing rapidly (summarized in (Avior et al., 2016; Sterneckert et al., 2014)).”

“…that a double strand break (DSB) generated by a SSN at a defined genomic site can be repaired either by the endogenous homology-mediated repair machinery using an exogenous provided repair template or by the error-prone Non-homologous end joining (NHEJ)-DNA repair pathway (Rouet et al., 1994a, b).”

“The low frequency of spontaneous homology mediated gene targeting in hPSCs was dramatically increased through the development of site-specific nucleases (SSN) as a tool for their genetic engineering (reviewed in (Carroll, 2014; Hsu et al., 2014; Urnov et al., 2010)).”

“Second, successful derivation of mice by SCNT with nuclei of B-cells as a donor, which had undergone VDJ-recombination, provided genetic evidence that terminally differentiated cells can be reprogrammed (Hochedlinger and Jaenisch, 2002).”

“…the SSN design, organism, and cell type and have already been to some extent implemented in hPSCs. Understanding the frequency and impact of off-targets is highly relevant to develop the use of SSN for clinical applications as well as for their reliable use in basic research (Gabriel et al., 2011).”

“, 2014) (Nelles et al., 2016), raising the possibly that it could be used to better understand the transcriptome in addition to the genome.”

“…have been successfully derived by fusions with functional protein that bind specific loci, or to activate or repress gene activity at the target site (Chen et al., 2013; Gilbert et al., 2014; Konermann et al., 2015; Mandegar et al., 2016; Tanenbaum et al., 2014) (CRISPRa and CRISPRi, Figure 2).”

“For example, this approach has been used successfully to identify the molecular principles underlying the most frequent non-coding mutations associated with human cancer (Bojesen et al., 2013; Fredriksson et al.; Horn et al., 2013; Huang et al., 2013; Killela et al., 2013).”

“…the use of SSNs such as ZFNs and transcription activator like effector nucleases (TALENs) to engineer hPSCs (DeKelver et al., 2010; Hockemeyer and Jaenisch, 2010; Hockemeyer et al., 2009; Hockemeyer et al., 2011; Lombardo et al., 2007; Sexton et al., 2014; Soldner et al., 2011; Zou et al., 2009).”

“For example, SSN-mediated correction of disease-causing mutations in LRKK2 that are associated in Parkinson’s disease revealed the transcriptional changes caused by disease-associated alleles in patient cells (Reinhardt et al., 2013).”

“As demonstrated for TALE proteins and Zinc finger DNA binding domains, the range of Cas9 could be extended in the future to also methylate or demethylate DNA or histones/chromatin at precise locations in the genome (Maeder et al., 2013; Meister et al., 2010).”

“A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript gene implicated in the pathogenesis of PD, by as little as 10% (Soldner et al, 2016).”

“For the mouse system it was essential to establish that iPSCs could pass the most stringent test for pluripotency: germ line transmission (Maherali et al., 2007; Okita et al., 2007; Wernig et al., 2007) and tetraploid complementation (Kang et al., 2009; Zhao et al., 2009).”

“In this system Cas9 expression can be induced by doxycycline so that efficient editing afterwards only requires the expression or delivery of the sgRNA (Gonzalez et al., 2014).”

“…challenge, recent studies used creative ways to take advantage of NHEJmeditated genome editing and the fact that the simultaneous expression of two nucleases can meditate the excision or inversion of the sequence internal to the two SSN (Chiba et al., 2015; (Chen et al., 2011; Young et al., 2016).”

“…(Chen et al., 2015; Cong et al., 2013; Fu et al., 2014; Gonzalez et al., 2014; Gu et al., 2015; Hou et al., 2013; Hsu et al., 2014; Kleinstiver et al., 2016; Liao and Karnik, 2015; Lin et al., 2014; Mali et al., 2013; Ran et al., 2015; Slaymaker et al., 2016; Tsai et al., 2014; Wu et al., 2014b).”

“An alternative protocol called BLES-seq that is based on directly labeling the DSBs generated by the nuclease in situ followed by enrichment through streptavidin affinity purification and next-generation sequencing (Crosetto et al., 2013) was originally developed to detect DSBs caused by replicative stress by stalled…”

“…state are at best small and overshadowed by differences caused by the reprograming method of choice, cell selection during propagation, culture conditions, and more importantly genetic background of the parental somatic cell (Guenther et al., 2010; (Kyttala et al., 2016); (Rouhani et al., 2014)).”

“Approaches to shift the balance away from NHEJ and towards homology-mediated repair included NHEJ inhibition with small molecules or by controlling the timing of CRISPR/Cas9 delivery with respect to the cell cycle stage (Chu et al., 2015; Maruyama et al., 2015; Robert et al., 2015; Yu et al., 2015).”

“While these data argue for the high specificity of Cas9, data in cancer cells suggest that offtargets can be frequently detected (Frock et al., 2015; Fu et al., 2014; Tsai et al., 2015; Wang et al., 2015b).”

“…loss-of-function screens, for example, to identify gene products that are required for drug resistance or to mediate viral cell death (Gilbert et al., 2014; Hart et al., 2015; Parnas et al., 2015; Shalem et al., 2014; Shi et al., 2015; Wang et al., 2015a; Wang et al., 2014a; Zhou et Cell Stem Cell.”

“Parallel differentiation of such isogenic sets of cells into disease-relevant cells and tissues can be used to directly assess the contribution of a mutation to cellular pathology (Chung et al., 2013; Ryan et al., 2013; Wang et al., 2014b; Yusa et al., 2011).”

“Similarly, two independent studies demonstrated the SSN can be used to directly correct b-thalassemia mutations in patient derived iPSCs and to restore hematopoietic differentiation (Ma et al., 2013; Xie et al., 2014).”

“…equally potent in reprogramming cells to a pluripotent state, providing valuable insights into the transcriptional pluripotency networks and how cells establish pluripotency (Buganim et al., 2012) (Apostolou and Hochedlinger, 2013; Park et al., 2008; Takahashi and Yamanaka, 2015; Yu et al., 2007).”

“…approach has been used to correct the disease-causing mutation with ornithine-daminotranferase that is mutated in patients with gyrate atrophy (Howden et al., 2011), or to alter the amount of disease-causing CAG repeat expansions in the huntingtin gene of patientspecific iPSCs (An et al., 2012).”

“…challenge, recent studies used creative ways to take advantage of NHEJmeditated genome editing and the fact that the simultaneous expression of two nucleases can meditate the excision or inversion of the sequence internal to the two SSN (Chiba et al., 2015; (Chen et al., 2011; Young et al., 2016).”

“…(Chen et al., 2015; Cong et al., 2013; Fu et al., 2014; Gonzalez et al., 2014; Gu et al., 2015; Hou et al., 2013; Hsu et al., 2014; Kleinstiver et al., 2016; Liao and Karnik, 2015; Lin et al., 2014; Mali et al., 2013; Ran et al., 2015; Slaymaker et al., 2016; Tsai et al., 2014; Wu et al., 2014b).”

“…generate animals carrying defined mutations in multiple species including fish, drosophila, mice and primates (Bassett et al., 2013; Chang et al., 2013; Gratz et al., 2013; Hwang et al., 2013a; Hwang et al., 2013b; Niu et al., 2014; Wang et al., 2013; Yang et al., 2013; Yu et al., 2013).”

“Similarly, single molecule imaging of Cas9 in living cells have demonstrated that Cas9 searches for target sites by three-dimensional diffusion, and that in contrast to on-target events off-target binding events are, on average, short-lived (<1 second) (Knight et al., 2015).”

“Using this approach Jiang et al. showed that ectopic expression of Xist was sufficient to transcriptionally suppress the targeted third copy of chromosome 21 and to reverse the cellular disease phenotypes in in vitro differentiated cells (Jiang et al., 2013).”

“Data form conventional whole genome sequencing of hPSCs exposed to Cas9 have so far been limited and not yet fully address the issues due to small sample sizes (Park et al., 2015; Smith et al., 2014).”

“…generate animals carrying defined mutations in multiple species including fish, drosophila, mice and primates (Bassett et al., 2013; Chang et al., 2013; Gratz et al., 2013; Hwang et al., 2013a; Hwang et al., 2013b; Niu et al., 2014; Wang et al., 2013; Yang et al., 2013; Yu et al., 2013).”

“…of patient-specific iPSCs followed by in vitro differentiation was also used to generate an isogenic disease model for cystic fibrosis by correcting disease-relevant mutations in CFTR followed by differentiation into airway epithelium (Crane et al., 2015; Firth et al., 2015; Suzuki et al., 2016).”

“…for Rett-syndrome disrupting MECP2 function in hPSCs (Li et al., 2013), to generate HIVresistant variants alleles of the CCR5 gene into iPSCs (Ye et al., 2014), to repair MYO15A in iPSCs derived from patients affected by deafness (Chen et al., 2016) and to derive isogeneic cell pairs of…”

“Protocols for conventional gene targeting have been optimized to modify human pluripotent stem cells (Costa et al., 2007; Davis et al., 2008a; Irion et al., 2007; Ruby and Zheng, 2009) and have been successfully used to establish hPSC models for human disease such as Lesch-Nyhan syndrome (Urbach et al.”

“…experiments, we and others implemented the use of SSNs such as ZFNs and transcription activator like effector nucleases (TALENs) to engineer hPSCs (DeKelver et al., 2010; Hockemeyer and Jaenisch, 2010; Hockemeyer et al., 2009; Hockemeyer et al., 2011; Lombardo et al., 2007; Sexton et al., 2014;…”

“Furthermore, genome editing in hPSCs has been used to establish models for Rett-syndrome disrupting MECP2 function in hPSCs (Li et al., 2013), to generate HIVresistant variants alleles of the CCR5 gene into iPSCs (Ye et al., 2014), to repair MYO15A in iPSCs derived from patients affected by…”

“Another challenge of genome editing in human cells is that human cells have relatively short conversion tracts (Elliott et al., 1998).”

“For one, it became evident that many protocols that were developed for the differentiation of hPSCs into functional tissue resulted in embryonic rather than adult human cell types (Bedada et al., 2015; Forster et al., 2014; Hrvatin et al., 2014; Spence et al., 2011; Takebe et al., 2013).”

“Data form conventional whole genome sequencing of hPSCs exposed to Cas9 have so far been limited and not yet fully address the issues due to small sample sizes (Park et al., 2015; Smith et al., 2014).”

“A similar approach to overcome the challenges associated with the low frequency of genecorrection events in hPSCs was used to correct point mutations in the beta-globin gene of iPSCs derived from patients with sickle cell disease (Zou et al., 2011).”

“…state are at best small and overshadowed by differences caused by the reprograming method of choice, cell selection during propagation, culture conditions, and more importantly genetic background of the parental somatic cell (Guenther et al., 2010; (Kyttala et al., 2016); (Rouhani et al., 2014)).”

“Alternative strategies for increasing editing efficiencies include methods to more efficiently detect and subclone cells that have undergone rare editing events (Miyaoka et al., 2014) as well as to enhance delivery methods for the nuclease and donor template (Lin et al.”

“, 2011) and Drosophila melanogaster (Beumer et al., 2008; Bibikova et al., 2003; Bibikova et al., 2002), which are similarly resilient to conventional gene-targeting strategies as hPSCs.”

“While technical challenges currently limit the potential application of such modifications, two recent papers describe gene editing of the embryo’s genome following injection of gRNAs, CRSPR/Cas9 RNA and targeting oligos into human zygotes (Kang et al., 2016; Liang et al., 2015).”

“…state are at best small and overshadowed by differences caused by the reprograming method of choice, cell selection during propagation, culture conditions, and more importantly genetic background of the parental somatic cell (Guenther et al., 2010; (Kyttala et al., 2016); (Rouhani et al., 2014)).”

“Approaches to shift the balance away from NHEJ and towards homology-mediated repair included NHEJ inhibition with small molecules or by controlling the timing of CRISPR/Cas9 delivery with respect to the cell cycle stage (Chu et al., 2015; Maruyama et al., 2015; Robert et al., 2015; Yu et al., 2015).”

“Facing this challenge, recent studies used creative ways to take advantage of NHEJmeditated genome editing and the fact that the simultaneous expression of two nucleases can meditate the excision or inversion of the sequence internal to the two SSN (Chiba et al., 2015; (Chen et al., 2011; Young et al., 2016).”

“Indeed, the number of human diseases modeled in culture using patient derived iPS cells (“disease in the dish”) is growing rapidly (summarized in (Avior et al., 2016; Sterneckert et al., 2014)).”

“Protocols for conventional gene targeting have been optimized to modify human pluripotent stem cells (Costa et al., 2007; Davis et al., 2008a; Irion et al., 2007; Ruby and Zheng, 2009) and have been successfully used to establish hPSC models for human disease such as Lesch-Nyhan syndrome (Urbach et…”

“, 2016) and to derive isogeneic cell pairs of COL7A1-corrected iPSCs derived from patients with dystrophic epidermolysis bullosa (Sebastiano et al., 2014).”

“, 2007; Ruby and Zheng, 2009) and have been successfully used to establish hPSC models for human disease such as Lesch-Nyhan syndrome (Urbach et al., 2004).”

“…of patient-specific iPSCs followed by in vitro differentiation was also used to generate an isogenic disease model for cystic fibrosis by correcting disease-relevant mutations in CFTR followed by differentiation into airway epithelium (Crane et al., 2015; Firth et al., 2015; Suzuki et al., 2016).”

“Though more challenging, SCNT was eventually successful in reprogramming human cells into hESCs in 2014 (Yamada et al., 2014).”

“One approach to increase the maturity of in vitro cell systems and to mimic cellular aging is to expose these cells to stressors that are associated with aging (Miller et al., 2013; Studer et al., 2015).”

“GWAS have identified genomic loci that may slightly increase the risk of developing a sporadic disease.”

“…to facilitate DNA-repair mediated editing of genomes proved to be almost universal and applies to hPSCs as well as other systems such as C. elegans (Morton et al., 2006; Wood et al., 2011) and Drosophila melanogaster (Beumer et al., 2008; Bibikova et al., 2003; Bibikova et al., 2002), which are…”

“…study risk alleles, patient-derived hiPSCs were used to dissect the effect of a common SAD-associated non-coding genetic variants in the 5′ region of the SORL1 (sortilin-related receptor, L(DLR class) A repeats containing) gene involved in intracellular vesicular trafficking (Young et al., 2015).”

“Parallel differentiation of such isogenic sets of cells into disease-relevant cells and tissues can be used to directly assess the contribution of a mutation to cellular pathology (Chung et al., 2013; Ryan et al., 2013; Wang et al., 2014b; Yusa et al., 2011).”

“This system has been used to generate loss of function alleles in EZH2 and to demonstrated the effects of haploinsufficeny for EZH2 in hematopoietic differentiation (Kotini et al., 2015).”

“Protocols for conventional gene targeting have been optimized to modify human pluripotent stem cells (Costa et al., 2007; Davis et al., 2008a; Irion et al., 2007; Ruby and Zheng, 2009) and have been successfully used to establish hPSC models for human disease such as Lesch-Nyhan syndrome (Urbach et al.”

“…became reality and Cas9-mediated genome engineering was developed into the platform of choice to generate SSNs and to genetically modify hPSCs (Chen et al., 2015; Cong et al., 2013; Fu et al., 2014; Gonzalez et al., 2014; Gu et al., 2015; Hou et al., 2013; Hsu et al., 2014; Kleinstiver et…”

“Similarly, two independent studies demonstrated the SSN can be used to directly correct b-thalassemia mutations in patient derived iPSCs and to restore hematopoietic differentiation (Ma et al., 2013; Xie et al., 2014).”

“Moreover, this approach has been used to correct the disease-causing mutation with ornithine-daminotranferase that is mutated in patients with gyrate atrophy (Howden et al., 2011), or to alter the amount of disease-causing CAG repeat expansions in the huntingtin gene of patientspecific iPSCs (An et al.”

“For one, it became evident that many protocols that were developed for the differentiation of hPSCs into functional tissue resulted in embryonic rather than adult human cell types (Bedada et al., 2015; Forster et al., 2014; Hrvatin et al., 2014; Spence et al., 2011; Takebe et al., 2013).”

“…equally potent in reprogramming cells to a pluripotent state, providing valuable insights into the transcriptional pluripotency networks and how cells establish pluripotency (Buganim et al., 2012) (Apostolou and Hochedlinger, 2013; Park et al., 2008; Takahashi and Yamanaka, 2015; Yu et al., 2007).”

“…the use of SSNs such as ZFNs and transcription activator like effector nucleases (TALENs) to engineer hPSCs (DeKelver et al., 2010; Hockemeyer and Jaenisch, 2010; Hockemeyer et al., 2009; Hockemeyer et al., 2011; Lombardo et al., 2007; Sexton et al., 2014; Soldner et al., 2011; Zou et al., 2009).”

“, 2014), to repair MYO15A in iPSCs derived from patients affected by deafness (Chen et al., 2016) and to derive isogeneic cell pairs of COL7A1-corrected iPSCs derived from patients with dystrophic epidermolysis bullosa (Sebastiano et al.”

“Howden et al demonstrated that human fibroblasts could be simultaneously reprogrammed and edited resulting in edited iPSCs going through only one single-cell cloning event without the need for drug selection (Howden et al., 2015).”

“Furthermore, several detailed protocols that describe the implementation of genome editing techniques in pluripotent stem cell systems have been optimized and published (Blair et al., 2016; Byrne and Church, 2015; Chiba and Hockemeyer, 2015; Yusa, 2013).”

“For one, it became evident that many protocols that were developed for the differentiation of hPSCs into functional tissue resulted in embryonic rather than adult human cell types (Bedada et al., 2015; Forster et al., 2014; Hrvatin et al., 2014; Spence et al., 2011; Takebe et al., 2013).”

“Decades before the study by Yamanaka, John Gurdon (Gurdon, 1962, 1963) had demonstrated that the epigenetic profile of a fully differentiated cell can be reprogrammed to a pluripotent state.”

“Furthermore, these protocols have been used to generate linage reporters for genes such as MIXL and Olig2 to study cell fate decision of differentiating human stem cells (Davis et al., 2008b; Xue et al., 2009).”

“Protocols for conventional gene targeting have been optimized to modify human pluripotent stem cells (Costa et al., 2007; Davis et al., 2008a; Irion et al., 2007; Ruby and Zheng, 2009) and have been successfully used to establish hPSC models for human disease such as Lesch-Nyhan syndrome (Urbach et al.”

“…implemented the use of SSNs such as ZFNs and transcription activator like effector nucleases (TALENs) to engineer hPSCs (DeKelver et al., 2010; Hockemeyer and Jaenisch, 2010; Hockemeyer et al., 2009; Hockemeyer et al., 2011; Lombardo et al., 2007; Sexton et al., 2014; Soldner et al., 2011;…”

“Protocols for conventional gene targeting have been optimized to modify human pluripotent stem cells (Costa et al., 2007; Davis et al., 2008a; Irion et al., 2007; Ruby and Zheng, 2009) and have been successfully used to establish hPSC models for human disease such as Lesch-Nyhan syndrome (Urbach et…”

“First, they demonstrated that the cytoplasm of an oocyte or an ESC contained diffusible transacting factors capable of reprogramming a somatic nucleus (reviewed in (Ambrosi and Rasmussen, 2005)).”

“…of patient-specific iPSCs followed by in vitro differentiation was also used to generate an isogenic disease model for cystic fibrosis by correcting disease-relevant mutations in CFTR followed by differentiation into airway epithelium (Crane et al., 2015; Firth et al., 2015; Suzuki et al., 2016).”

“As demonstrated for TALE proteins and Zinc finger DNA binding domains, the range of Cas9 could be extended in the future to also methylate or demethylate DNA or histones/chromatin at precise locations in the genome (Maeder et al., 2013; Meister et al., 2010).”

“Combining the cellular versatility of iPSC differentiation with the ease of CRISPR/Cas9-mediated genome editing proved to be a very powerful experimental approach and by now genome editing in hPSCs has become a standard tool in stem cell research and human disease modeling (Johnson and Hockemeyer, 2015; Matano et al.,…”

“Furthermore, several detailed protocols that describe the implementation of genome editing techniques in pluripotent stem cell systems have been optimized and published (Blair et al., 2016; Byrne and Church, 2015; Chiba and Hockemeyer, 2015; Yusa, 2013).”

“Furthermore, several detailed protocols that describe the implementation of genome editing techniques in pluripotent stem cell systems have been optimized and published (Blair et al., 2016; Byrne and Church, 2015; Chiba and Hockemeyer, 2015; Yusa, 2013).”

“Furthermore, several detailed protocols that describe the implementation of genome editing techniques in pluripotent stem cell systems have been optimized and published (Blair et al., 2016; Byrne and Church, 2015; Chiba and Hockemeyer, 2015; Yusa, 2013).”

“Indeed, the efficiency of altering the genome of mammals by injecting CRISPR/Cas9 RNA or DNA into the fertilized egg (Wang et al, 2013) sparked a debate whether this technology should be used to modify the human germ line (Sheridan, 2015).”

“Efforts such as the one launched by the Allen Institute for Cell Science to create an industrial scale library of characterized iPSCs that will be used to create a visual, animated model of the cell, suggest that iPSCs will soon replace cancer cells as a model system for basic cell biology (Callaway, 2014).”

“Use of CRISPR technology in iPSCs (Hockemeyer and Jaenisch 2016).”

“The combined use of iPSCs and genome editing technologies has enabled revolutionary advances in many areas (61).”

“The generation of isogenic iPSC lines using genome-editing technologies, with the edited gene as the sole variable, allows the identification of pathological phenotypes that are specific to the genetic risk factors, without potential confusion resulting from distinct genetic or epigenetic background of different iPSC…”

“In addition, iPSCs can be reprogrammed from patient cells via small molecules, miRNAs, and combinations of reprogramming factors, and be differentiated into somatic cells for drug testing and regenerative medicine.(179) However, many studies clarified that ageing also might constitute critical barriers to cell reprogr…”

“With rapid development of genome editing technologies, the generation of isogenic iPSC lines with the edited gene as the sole variable allows for identification of true pathological phenotypes, without worrying about changes resulting from different genetic or epigenetic backgrounds (Hockemeyer and Jaenisch, 2016).”

“The technology has already shown promise for developing treatments and potentially curing some inherited genetic conditions (Hockemeyer and Jaenisch, 2016).”

“…theoretical capacity to differentiate into essentially every cell type, and the potential to model a pathogenic genotype by reprogramming cells from an affected individual, iPSCs have attracted considerable attention (Soldner et al., 2012; Sterneckert et al., 2014; Hockemeyer and Jaenisch, 2016).”

“Gene targeting of PSCs is broadly relevant for realizing the full potential of these applications 73 .”

“113,114 The aforementioned gene-editing can be used to restore the effect of specific genes or mutations while exploring genotypic effects on specific phenotypes.”

“Such effects may be specific to cell types and/or specific developmental stages (Duan, 2015; Hockemeyer and Jaenisch, 2016).”

“We apologize to those whose findings are relevant but not cited in this review due to space limitation.”

“In addition, powerful genome editing tools such as the CRISPR/Cas9 system enable scientists to freely modify the genetic information of hPSC (Hockemeyer and Jaenisch, 2016; Kim et al., 2019b).”

“…CRISPR-Cas9 by correcting the disease-causing mutation in a patient hiPSC line or introducing it in a control line, thus generating pairs of cell lines that are identical in their genetic background and differ only by the presence or absence of a specific mutation (Hockemeyer & Jaenisch 2016)).”

“Additionally, recent advancements in genome-editing strategies by CRISPR/Cas9 technology have greatly expanded the potential of investigation of iPSC-based models, allowing the generation of isogenic iPSC lines, in which a desired gene mutation is either introduced or corrected, and the development of model systems th…”

“When researchers want to edit cells, they usually edit stem cells, which can be maintained in culture indefinitely and (perhaps) differentiated into other cell types as required (Hockemeyer and Jaenisch 2016).”

“Such ‘‘engineering in’’ of disease-associated mutations into a putatively WT or normal cell line is desirable to create sets of isogenic cell lines for rigorous study of genotype-phenotype relationships (Hockemeyer and Jaenisch, 2016; Merkle and Eggan, 2013).”

“However, recent advances in in vitro stem cell culture and haematopoietic lineage differentiation, as well as advances in genome editing, offer exciting perspectives to address a number of these issues in the near future (Hockemeyer and Jaenisch, 2016; Caulier et al., 2017).”

“One of the current challenges of using human pluripotent stem cells (hPSCs) to model human diseases is to precisely and efficiently engineer the genome to introduce designer mutations ( Hockemeyer and Jaenisch, 2016 ; Soldner and Jaenisch, 2018 ).”

“84 Inspiring examples to this effect include work by Dirk Hockemeyer on control of telomere homeiostasis, 85 and Danwei Huangfu on epigenome maintenance.”

“To evaluate homology-directed repair (HDR) effectiveness, we examined point mutation frequencies in the window and its flanking regions, and categorized the sequences into four groups: (1) ‘Perfect HDR’: the repair has all”

“Neurological Disorders The parallel development of iPSC and gene editing technologies has been serendipitous for advancing the understanding of neurological disorders (Figure 2) (Hockemeyer and Jaenisch, 2017).”

“Many approaches have been employed to improve the odds of achieving the intended edit and reduce the burden of screening hundreds of clones (Blair et al., 2016; Hockemeyer and Jaenisch, 2016; Ikeda et al., 2018; Kwart et al., 2017; Merkle et al., 2015; Popp et al., 2018; Steyer et al., 2018).”

“…patients’ fibroblasts can be used for disease modeling, comparison of organoids derived from patient and control iPS cell lines may not reveal disease-relevant phenotypes, but rather may reflect the variation in the genetic backgrounds and the reprogramming history of individual cell lines [24].”

“Genome-engineering and chromatin-modifying techniques in pluripotent stem cells and their progeny in 2D and 3D cultures are continuing to improve in both effectiveness and practicality (Hockemeyer and Jaenisch, 2016; Pulecio et al., 2017; Shi et al., 2017).”

“The guide RNA sequences are simple to design and directly target specific genes 83 .”

“The latter studies are performed using patient-derived iPSCs that are differentiated into a plethora of brain cells, including neurons, astrocytes, oligodendrocytes, and recently microglia, systems that were previously not readily available for experimental investigation (Hockemeyer and Jaenisch, 2016; Soldner et al.,…”

“In the last few years, the development of CRISPR/ Cas9 technology has made it much easier to manipulate the genome of human cells (Hockemeyer and Jaenisch, 2016).”

“Sequence insertions are often used for the generation of cell type-specific reporter alleles or the ectopic expression of therapeutic proteins [7] [8].”

“…in genome editing technologies enable the correction of a pathogenic mutation in patient iPSCs or the introduction of a desired genetic alteration in healthy iPSCs for studying genetic function or modulating cellular characteristics (Hockemeyer and Jaenisch, 2016; Hotta and Yamanaka, 2015).”