Genome-engineering with CRISPR-Cas9 in the mosquito Aedes aegypti

Article summary

The mosquito Aedes aegypti is a potent vector of the Chikungunya, yellow fever, and Dengue viruses, which result in hundreds of millions of infections and over 50,000 human deaths per year. Loss-of-function mutagenesis in Ae. aegypti has been established with TALENs, ZFNs, and homing endonucleases, which require the engineering of DNA-binding protein domains to generate target specificity for a particular stretch of genomic DNA. Here, we describe the first use of the CRISPR-Cas9 system to generate targeted, site-specific mutations in Ae. aegypti. CRISPR-Cas9 relies on RNA-DNA base-pairing to generate targeting specificity, resulting in cheaper, faster, and more flexible genome-editing reagents. We investigate the efficiency of reagent concentrations and compositions, demonstrate the ability of CRISPR-Cas9 to generate several different types of mutations via disparate repair mechanisms, and show that stable germ-line mutations can be readily generated at the vast majority of genomic loci tested. This work offers a detailed exploration into the optimal use of CRISPR-Cas9 in Ae. aegypti that should be applicable to non-model organisms previously out of reach of genetic modification.

Reference: Kistler et al Genome-engineering with CRISPR-Cas9 in the mosquito Aedes aegypti Here

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An Active Immune Defence with a Minimal CRISPR (clustered regularly interspaced short palindromic repeats) RNA and Without the Cas6 Protein

Article summary

The prokaryotic immune system CRISPR-Cas1 is a defence system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III crRNAs are generated by the endonuclease Cas6. We developed a Cas6b2-independent crRNA maturation pathway for the Haloferax type I-B system in vivo, that expresses a functional crRNA that we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analysed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3′ handle are still active in triggering a interference reaction. The complete 3′ handle could be removed without loss of activity. However manipulations of the 5′ handle mostly led to loss of interference activity. Furthermore we could show that in the presence of an icrRNA a strain without Cas6b (∆cas6b) is still active in interference.

Reference:Maier et al J. Biol. Chem. jbc.M114.617506. doi:10.1074/jbc.M114.617506

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Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery

Article summary

The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.

Reference:Lin et al eLife 2014;10.7554/eLife.04766

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Site-directed mutagenesis in Arabidopsis thaliana using dividing tissue-targeted RGEN of the CRISPR/Cas system to generate heritable null alleles

Article summary

Site-directed genome engineering in higher plants has great potential for basic research and molecular breeding. Here, we describe a method for site-directed mutagenesis of the Arabidopsis nuclear genome that efficiently generates heritable mutations using the RNA-guided endonuclease (RGEN) derived from bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 (CRISPR associated) protein system. To induce mutagenesis in proliferating tissues during embryogenesis and throughout the plant life cycle, the single guide RNA (sgRNA) and Cas9 DNA endonuclease were expressed from the U6 snRNA and INCURVATA2 promoters, respectively. After Agrobacterium-mediated introduction of T-DNAs encoding RGENs that targets FLOWERING LOCUS T (FT) and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 4 genes, somatic mutagenesis at the targeted loci was observed in T1 transformants. In the results of FT-RGEN, T1 plants often showed late flowering indicative of the presence of large somatic sectors in which the FT gene is mutated on both chromosomes. DNA sequencing analysis estimated that about 90 % of independent chromosomal DNA fragments carried mutations in the analyzed tissue of a T1 plant showing late flowering. The most frequently detected somatic polymorphism showed a high rate of inheritance in T2 plants, and inheritance of less frequent polymorphisms was also observed. As a result, late-flowering plants homozygous for novel, heritable null alleles of FT including a 1 bp insertion or short deletions were recovered in the following T2 and T3 generations. Our results demonstrate that dividing tissue-targeted mutagenesis using RGEN provides an efficient heritable genome engineering method in A. thaliana.

Reference:Hyun, Youbong, et al. "Site-directed mutagenesis in Arabidopsis thaliana using dividing tissue-targeted RGEN of the CRISPR/Cas system to generate heritable null alleles." Planta (2014): 1-14.

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Generation of WNK1 knockout cell lines by CRISPR/Cas-mediated genome editing

Article Abstract

Sodium-coupled SLC12 cation chloride cotransporters play important roles in cell volume and chloride homeostasis, epithelial fluid secretion, and renal tubular salt reabsorption. These cotransporters are phosphorylated and activated indirectly by With-No-Lysine (WNK) kinases through their downstream effector kinases, SPAK and OSR1. Multiple WNK kinases can coexist within a single cell type, although their relative contributions to SPAK/OSR1 activation and salt transport remain incompletely understood. Deletion of specific WNKs from cells that natively express a functional WNK-SPAK/OSR1 network will help resolve these knowledge gaps. Here, we outline a simple method to selectively knock out full length WNK1 expression from mammalian cells using RNA-guided CRISPR/Cas9 endonucleases. Two clonal cell lines were generated by using a single guide RNA (sgRNA) targeting exon 1 of the WNK1 gene, which produced indels that abolished WNK1 protein expression. Both cell lines exhibited reduced endogenous WNK4 protein abundance, indicating that WNK1 is required for WNK4 stability. Consistent with an on-target effect, the reduced WNK4 abundance was associated with increased expression of the KLHL3/Cullin-3 E3 ubiquitin ligase complex, and was rescued by exogenous WNK1 overexpression. Although the morphology of the knockout cells was indistinguishable from control, they exhibited low baseline SPAK/OSR1 activity and failed to trigger regulatory volume increase (RVI) following hypertonic stress, confirming an essential role for WNK1 in cell volume regulation. Collectively, our data show how this new, powerful, and accessible gene editing technology can be used to dissect and analyze WNK signaling networks.

Reference: Roy, Ankita, et al. "Generation of WNK1 knockout cell lines by CRISPR/Cas-mediated genome editing." American Journal of Physiology-Renal Physiology (2014): ajprenal-00612.

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Optimization of scarless human stem cell genome editing

Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. Here, authors present several improvements: First, they developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. Then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. Authors found Cas9-gRNA achieved 7–8× higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design, they demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks.

Reference:Yang, Luhan, et al. "Optimization of scarless human stem cell genome editing." Nucleic acids research 41.19 (2013): 9049-9061.

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The CRISPR–Cas system for plant genome editing: advances and opportunities

Genome editing is an approach in which a specific target DNA sequence of the genome is altered by adding, removing, or replacing DNA bases. Artificially engineered hybrid enzymes, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), and the CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated protein) system are being used for genome editing in various organisms including plants. The CRISPR–Cas system has been developed most recently and seems to be more efficient and less time-consuming compared with ZFNs or TALENs. This system employs an RNA-guided nuclease, Cas9, to induce double-strand breaks. The Cas9-mediated breaks are repaired by cellular DNA repair mechanisms and mediate gene/genome modifications. Here, authors provide a detailed overview of the CRISPR–Cas system and its adoption in different organisms, especially plants, for various applications. Important considerations and future opportunities for deployment of the CRISPR–Cas system in plants for numerous applications are also discussed. Recent investigations have revealed the implications of the CRISPR–Cas system as a promising tool for targeted genetic modifications in plants. This technology is likely to be more commonly adopted in plant functional genomics studies and crop improvement in the near future.

Reference: Vinay Kumar and Mukesh Jain. The CRISPR–Cas system for plant genome editing: advances and opportunities J. Exp. Bot. first published online November 4, 2014 doi:10.1093/jxb/eru429

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Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9

The CRISPR/Cas9 system has ushered in a new era of targeted genetic manipulations. Here, authors report the use of CRISPR/Cas9 to induce double-stranded breaks in the genome of the sea squirt Ciona intestinalis. They use electroporation to deliver CRISPR/Cas9 components for tissue-specific disruption of the Ebf (Collier/Olf/EBF) gene in hundreds of synchronized Ciona embryos. Phenotyping of transfected embryos in the ‘F0’ generation revealed that endogenous Ebf function is required for specification of Islet-expressing motor ganglion neurons and atrial siphon muscles. They demonstrate that CRISPR/Cas9 is sufficiently effective and specific to generate large numbers of embryos carrying mutations in a targeted gene of interest, which should allow for rapid screening of gene function in Ciona.

Reference:Stolfi, Alberto, et al. "Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9." Development 141.21 (2014): 4115-4120.

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Enhanced Specificity and Efficiency of the CRISPR/Cas9 System with Optimized sgRNA Parameters in Drosophila

Summary

The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in Drosophila melanogaster. However, single-guide RNA (sgRNA) parameters affecting the specificity and efficiency of the system in flies are still not clear. Here, authors found that off-target effects did not occur in regions of genomic DNA with three or more nucleotide mismatches to sgRNAs. Importantly, they document for a strong positive correlation between mutagenesis efficiency and sgRNA GC content of the six protospacer-adjacent motif-proximal nucleotides (PAMPNs). Furthermore, by injecting well-designed sgRNA plasmids at the optimal concentration they determined, authors could efficiently generate mutations in four genes in one step. Finally, they generated null alleles of HP1a using optimized parameters through homology-directed repair and achieved an overall mutagenesis rate significantly higher than previously reported. This work demonstrates a comprehensive optimization of sgRNA and promises to vastly simplify CRISPR/Cas9 experiments in Drosophila.

Reference:Ren et al., 2014, Enhanced Specificity and Efficiency of the CRISPR/Cas9 System with Optimized sgRNA Parameters in Drosophila. Cell Reports 9, 1–12

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A CRISPR/Cas9-Based System for Reprogramming Cell Lineage Specification

Summary

Gene activation by the CRISPR/Cas9 system has the potential to enable new approaches to science and medicine, but the technology must be enhanced to robustly control cell behavior. Authors show that the fusion of two transactivation domains to Cas9 dramatically enhances gene activation to a level that is necessary to reprogram cell phenotype. Targeted activation of the endogenous Myod1 gene locus with this system led to stable and sustained reprogramming of mouse embryonic fibroblasts into skeletal myocytes. The levels of myogenic marker expression obtained by the activation of endogenous Myod1 gene were comparable to that achieved by overexpression of lentivirally delivered MYOD1 transcription factor.

Reference:Chakraborty et al., A CRISPR/Cas9-Based System for Reprogramming Cell Lineage Specification, Stem Cell Reports (2014), http://dx.doi.org/10.1016/j.stemcr.2014.09.013

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MacSyFinder: A Program to Mine Genomes for Molecular Systems with an Application to CRISPR-Cas Systems

Motivation

 Biologists often wish to use their knowledge on a few experimental models of a given molecular system to identify homologs in genomic data. We developed a generic tool for this purpose. 

 Results

 Macromolecular System Finder (MacSyFinder) provides a flexible framework to model the properties of molecular systems (cellular machinery or pathway) including their components, evolutionary associations with other systems and genetic architecture. Modelled features also include functional analogs, and the multiple uses of a same component by different systems. Models are used to search for molecular systems in complete genomes or in unstructured data like metagenomes. The components of the systems are searched by sequence similarity using Hidden Markov model (HMM) protein profiles. The assignment of hits to a given system is decided based on compliance with the content and organization of the system model. A graphical interface, MacSyView, facilitates the analysis of the results by showing overviews of component content and genomic context. To exemplify the use of MacSyFinder we built models to detect and class CRISPR-Cas systems following a previously established classification. Authors show that MacSyFinder allows to easily define an accurate “Cas-finder” using publicly available protein profiles. 

 Availability and Implementation 

 MacSyFinder is a standalone application implemented in Python. It requires Python 2.7, Hmmer and makeblastdb (version 2.2.28 or higher). It is freely available with its source code under a GPLv3 license at https://github.com/gem-pasteur/macsyfind​er. It is compatible with all platforms supporting Python and Hmmer/makeblastdb. The “Cas-finder” (models and HMM profiles) is distributed as a compressed tarball archive as Supporting Information.

Reference:

Abby SS, Néron B, Ménager H, Touchon M, Rocha EPC (2014) MacSyFinder: A Program to Mine Genomes for Molecular Systems with an Application to CRISPR-Cas Systems. PLoS ONE 9(10): e110726. doi:10.1371/journal.pone.0110726

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Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation

While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. Authors present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. They identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%–99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, researchers construct genome-scale CRISPRi and CRISPRa libraries, each of which they validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. These results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.

Reference:Luke A. Gilbert, Max A. Horlbeck, Britt Adamson, Jacqueline E. Villalta, Yuwen Chen, Evan H. Whitehead, Carla Guimaraes, Barbara Panning, Hidde L. Ploegh, Michael C. Bassik, Lei S. Qi, Martin Kampmann, Jonathan S. Weissman, Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation, Cell, Available online 9 October 2014, ISSN 0092-8674, http://dx.doi.org/10.1016/j.cell.2014.09.029.

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Characterization of genomic deletion efficiency mediated by clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells

The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, Authors present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. They observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. Authors retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation.

Reference:Ran, F., et al. "Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity." Cell 154.6 (2013): 1380-1389.

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Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR-Cas9 system

Cancer-related human chromosomal translocations are generated through the illegitimate joining of two non-homologous chromosomes affected by double-strand breaks (DSB). Effective methodologies to reproduce precise reciprocal tumour-associated chromosomal translocations are required to gain insight into the initiation of leukaemia and sarcomas. Here researchers present a strategy for generating cancer-related human chromosomal translocations in vitro based on the ability of the RNA-guided CRISPR-Cas9 system to induce DSBs at defined positions. Using this approach authors generate human cell lines and primary cells bearing chromosomal translocations resembling those described in acute myeloid leukaemia and Ewing's sarcoma at high frequencies. FISH and molecular analysis at the mRNA and protein levels of the fusion genes involved in these engineered cells reveal the reliability and accuracy of the CRISPR-Cas9 approach, providing a powerful tool for cancer studies.

Reference:Torres, R., et al. "Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR–Cas9 system." Nature Communications 5 (2014).

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Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system.

Introducing a point mutation is a fundamental method used to demonstrate the roles of particular nucleotides or amino acids in the genetic elements or proteins, and is widely used in in vitro experiments based on cultured cells and exogenously provided DNA. However, the in vivo application of this approach by modifying genomic loci is uncommon, partly due to its technical and temporal demands. This leaves many in vitro findings un-validated under in vivo conditions. Authors herein applied the CRISPR/Cas9 system to generate mice with point mutations in their genomes, which led to single amino acid substitutions in proteins of interest. By microinjecting gRNA, hCas9 mRNA and single-stranded donor oligonucleotides (ssODN) into mouse zygotes, researchers introduced defined genomic modifications in their genome with a low cost and in a short time. Both single gRNA/WT hCas9 and double nicking set-ups were effective. They also found that the distance between the modification site and gRNA target site was a significant parameter affecting the efficiency of the substitution. Authors believe that this is a powerful technique that can be used to examine the relevance of in vitro findings, as well as the mutations found in patients with genetic disorders, in an in vivo system.

Reference:Inui, Masafumi, et al. "Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system." Scientific reports 4 (2014).

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Whole-Genome Sequencing Analysis Reveals High Specificity of CRISPR/Cas9 and TALEN-Based Genome Editing in Human iPSCs

A lettter is published by Smith et al about CRISPR and TALEN-Based Genome Editing in Human iPSCs

Reference:Smith, Cory, et al. "Whole-Genome Sequencing Analysis Reveals High Specificity of CRISPR/Cas9 and TALEN-Based Genome Editing in Human iPSCs." Cell stem cell 15.1 (2014): 12-13.

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Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing.

Genome editing has attracted wide interest for the generation of cellular models of disease using human pluripotent stem cells and other cell types. CRISPR-Cas systems and TALENs can target desired genomic sites with high efficiency in human cells, but recent publications have led to concern about the extent to which these tools may cause off-target mutagenic effects that could potentially confound disease-modeling studies. Using CRISPR-Cas9 and TALEN targeted human pluripotent stem cell clones, authors performed whole-genome sequencing at high coverage in order to assess the degree of mutagenesis across the entire genome. In both types of clones, authors found that off-target mutations attributable to the nucleases were very rare. From this analysis, researchers suggest that, although some cell types may be at risk for off-target mutations, the incidence of such effects in human pluripotent stem cells may be sufficiently low and thus not a significant concern for disease modeling and other applications.

Reference:Veres, Adrian, et al. "Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing." Cell stem cell 15.1 (2014): 27-30.

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Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes.

Tyrosinase is the rate-limiting enzyme for the production of melanin pigmentation. In the mouse and other animals, homozygous null mutations in the Tyrosinase gene (Tyr) result in the absence of pigmentation, i.e. albinism. Here authors used the CRISPR/Cas9 system to generate mono- and bi-allelic null mutations in the Tyr locus by zygote injection of two single-guide and Cas9 RNAs. Injection into C57BL/6N wild-type embryos resulted in one completely albino founder carrying two different Tyr mutations. In addition, three pigmentation mosaics and fully pigmented littermates were obtained that transmitted new mutant Tyr alleles to progeny in test crosses with albinos. Injection into Tyr heterozygous (B6CBAF1/J×FVB/NJ) zygotes resulted in the generation of numerous albinos and also mice with a graded range of albino mosaicism. Deep sequencing revealed that the majority of the albinos and the mosaics had more than two new mutant alleles. These visual phenotypes and molecular genotypes highlight the somatic mosaicism and allele complexity in founders that occurs for targeted genes during CRISPR/Cas9-mediated mutagenesis by zygote injection in mice.

Reference:Yen, Shuo-Ting, et al. "Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes." Developmental biology 393.1 (2014): 3-9.

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Generation of eGFP and Cre knockin rats by CRISPR/Cas9

The type II bacterial CRISPR/Cas [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)] system is a very valuable genome engineering tool, which has been widely used in genome editing of a variety of organisms. Previously, we generated floxed alleles in rats by CRISPR/Cas9. Here, authors successfully use a two-cut strategy with one circular vector, which contains the exogenous cDNAs with homology arm regions, in generating knockin rats at the Trdmt1, Nestin and Cck loci. The efficiency of CRISPR/Cas9-mediated knockin was up to 54%. Furthermore, by crossing the Nestin-Cre rat with the Dnmt3b floxed rat and Cck-Cre with the Dnmt1 floxed rat, they detected Cre/loxP-mediated recombination in the F1 generation of rats. They also show that the knockin alleles were germline transmitted. These results provided a simple and flexible engineering strategy for the establishment of knockin rats.

Reference:Ma, Yuanwu, et al. "Generation of eGFP and Cre knockin rats by CRISPR/Cas9." FEBS Journal 281.17 (2014): 3779-3790.

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Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac

β-thalassemia, one of the most common genetic diseases worldwide, is caused by mutations in the human hemoglobin beta (HBB) gene. Creation of human induced pluripotent stem cells (iPSCs) from β-thalassemia patients could offer an approach to cure this disease. Correction of the disease-causing mutations in iPSCs could restore normal function and provide a rich source of cells for transplantation. In this study, authors used the latest gene-editing tool, CRISPR/Cas9 technology, combined with the piggyBac transposon to efficiently correct the HBB mutations in patient-derived iPSCs without leaving any residual footprint. No off-target effects were detected in the corrected iPSCs, and the cells retain full pluripotency and exhibit normal karyotypes. When differentiated into erythroblasts using a monolayer culture, gene-corrected iPSCs restored expression of HBB compared to the parental iPSCs line. This study provides an effective approach to correct HBB mutations without leaving any genetic footprint in patient-derived iPSCs, thereby demonstrating a critical step toward the future application of stem cell-based gene therapy to monogenic diseases.

Reference:Xie, Fei, et al. "Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac." Genome research 24.9 (2014): 1526-1533.

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