Next-generation CRISPR technology for genome, epigenome and mitochondrial editing.

The application of rapidly growing CRISPR toolboxes and methods has great potential to transform biomedical research. Here, we provide a snapshot of up-to-date CRISPR toolboxes, then critically discuss the promises and hurdles associated with CRISPR-based nuclear genome editing, epigenome editing, and mitochondrial editing. The technical challenges and key solutions to realize epigenome editing in vivo, in vivo base editing and prime editing, mitochondrial editing in complex tissues and animals, and CRISPR-associated transposases and integrases in targeted genomic integration of very large DNA payloads are discussed. Lastly, we discuss the latest situation of the CRISPR/Cas9 clinical trials and provide perspectives on CRISPR-based gene therapy. Apart from technical shortcomings, ethical and societal considerations for CRISPR applications in human therapeutics and research are extensively highlighted.

Development of a baculoviral CRISPR/Cas9 vector system for beta-2-microglobulin knockout in human pluripotent stem cells.

Derivation of hypoimmunogenic human cells from genetically manipulated pluripotent stem cells holds great promise for future transplantation medicine and adoptive immunotherapy. Disruption of beta-2-microglobulin (B2M) in pluripotent stem cells followed by differentiation into specialized cell types is a promising approach to derive hypoimmunogenic cells. Given the attractive features of CRISPR/Cas9-based gene editing tool and baculoviral delivery system, baculovirus can deliver CRISPR/Cas9 components for site-specific gene editing of B2M. Herein, we report the development of a baculoviral CRISPR/Cas9 vector system for the B2M locus disruption in human cells. When tested in human embryonic stem cells (hESCs), the B2M gene knockdown/out was successfully achieved, leading to the stable down-regulation of human leukocyte antigen class I expression on the cell surface. Fibroblasts derived from the B2M gene-disrupted hESCs were then used as stimulator cells in the co-cultures with human peripheral blood mononuclear cells. These fibroblasts triggered significantly reduced alloimmune responses as assessed by sensitive Elispot assays. The B2M-negative hESCs maintained the pluripotency and the ability to differentiate into three germ lineages in vitro and in vivo. These findings demonstrated the feasibility of using the baculoviral-CRISPR/Cas9 system to establish B2M-disrupted pluripotent stem cells. B2M knockdown/out sufficiently leads to hypoimmunogenic conditions, thereby supporting the potential use of B2M-negative cells as universal donor cells for allogeneic cell therapy.

Rapid and Amplification-free Nucleic Acid Detection with DNA Substrate-Mediated Autocatalysis of CRISPR/Cas12a.

To enable rapid and accurate point-of-care DNA detection, we have developed a single-step, amplification-free nucleic acid detection platform, a DNA substrate-mediated autocatalysis of CRISPR/Cas12a (DSAC). DSAC makes use of the trans-cleavage activity of Cas12a and target template-activated DNA substrate for dual signal amplifications. DSAC employs two distinct DNA substrate types: one that enhances signal amplification and the other that negatively modulates fluorescent signals. The positive inducer utilizes nicked- or loop-based DNA substrates to activate CRISPR/Cas12a, initiating trans-cleavage activity in a positive feedback loop, ultimately amplifying the fluorescent signals. The negative modulator, which involves competitor-based DNA substrates, competes with the probes for trans-cleaving, resulting in a signal decline in the presence of target DNA. These DNA substrate-based DSAC systems were adapted to fluorescence-based and paper-based lateral flow strip detection platforms. Our DSAC system accurately detected African swine fever virus (ASFV) in swine's blood samples at femtomolar sensitivity within 20 min. In contrast to the existing amplification-free CRISPR/Dx platforms, DSAC offers a cost-effective and straightforward detection method, requiring only the addition of a rationally designed DNA oligonucleotide. Notably, a common ASFV sequence-encoded DNA substrate can be directly applied to detect human nucleic acids through a dual crRNA targeting system. Consequently, our single-step DSAC system presents an alternative point-of-care diagnostic tool for the sensitive, accurate, and timely diagnosis of viral infections with potential applicability to human disease detection.

Relationship Between Genetic Polymorphism and Cognitive Impairment in Patients with Acute Ischemic Stroke (APOE).

Objective: To investigate the relationship between apolipoprotein E (ApoE) gene polymorphism and cognitive impairment (PSCI) in patients after acute ischemic stroke (AIS). Methods: A total of 150 AIS patients were treated in Chengde Central Hospital from December 2022 to December 2023 and were selected and divided into a disorder group (n=88) and a normal group (n=62) according to the presence or absence of PSCI. Clinical data of patients in the two groups were collected, ApoE genotype and allele distribution of patients in the disabled group and the normal group were detected, Montreal Cognitive Assessment and Mini-Mental State Examination scores of patients with different ApoE gene subtypes were compared, and the risk factors of PSCI after AIS were analyzed by unconditional Logistic regression. Results: The proportion of patients with acute lesions (≥3.0 cm) and the degree of carotid artery stenosis (moderate, severe, complete occlusion) in the disorder group was higher than that in the normal group, and the National Institutes of Health Stroke Scale score was higher than that in the control group, with statistical significance (P < .05). There were significant differences in the genotype and allelic distribution of ApoE between the two groups (P < .05). In both groups, the highest genotype frequency of ApoE was the ε3/3 homozygous type, which was 47.73% (in the disorder group) and 72.58% (in the normal group) respectively. In contrast, there were no significant differences in the genotype frequencies of ε2/2, ε2/3, ε2/4 and ε4/4 alleles in the two groups (P > .05). This means that in both groups of patients, the frequency of the ApoE ε3/3 genotype was the highest, while the genotype frequencies of ε2/2, ε2/3, ε2/4 and ε4/4 alleles were not significant between the two groups. difference. The distribution differences of these genotypes and alleles may be related to aspects such as disease risk and physiological function, providing valuable information for in-depth exploration of the role of ApoE in patients. The genotype frequency of ε3/3 in the disorder group was lower than that in the normal group. The frequency of the ε3/4 genotype was higher than that of the normal group, and the difference was statistically significant (P < .05). In both groups, the highest allele frequency was ε3 (68.75% in the disorder group and 83.06% in the normal group), and there was no difference in the frequency of ε2 allele between the two groups (P > .05). The frequency of the ε3 allele in the disorder group was lower than that in the normal group, and the frequency of the ε4 allele was higher than that in the normal group, the difference was statistically significant (P < .05). In the patients with cognitive impairment after AIS (disorder group), the MOCA and MMSE scores of patients with different ApoE subtypes (ε2, ε3, ε4) were compared, and the differences among the three groups were statistically significant (P < .05). The MOCA and MMSE scores in the ε4 group were lower than those in the ε2 and ε3 groups. The difference was statistically significant (P < .05). Logistic regression analysis showed that the degree of carotid artery stenosis, NIHSS score, and ApoEε4 gene were independent risk factors for PSCI in patients with AIS (P < .05). Conclusion: APOE gene polymorphism is associated with cognitive impairment in post-AIS patients, and carrying the ApoE Epsilon 4 gene may be associated with PSCI in post-AIS patients.

Trace determination of disinfection by-products in drinking water by cyclic ion chromatography with large-volume direct injection.

A novel cyclic ion chromatography (IC) system was developed for the simultaneous determination of trace disinfection by-products (DBPs) in drinking water. Five DBPs (chlorite, bromate, chlorate, dichloroacetic acid, and trichloroacetic acid) were sensitively determined by large-volume direct injection, and the interferences of dominant inorganic anions present in water were eliminated online through the cyclic determination of the target analytes. Under optimized conditions, the obtained limits of detection (LODs) were in the range of 0.18-1.91 µg L-1 based on a signal-to-noise ratio (S/N) of 3 and an injection volume of 1.0 mL. The RSDs for peak area and retention time were in the range of 0.13-1.03% and 1.24-4.29%, respectively. Satisfactory recoveries between 92.3% and 106.4% were obtained by adding three concentration gradients of standards to the drinking water samples. The proposed method has advantages such as high sensitivity, facile automation, and no sample pretreatment, and might be a promising approach for routine analysis.

Occurrence, seasonal variations, distribution patterns, and risk assessment of volatile monoaromatic hydrocarbons in soils of industrial parks in Yangtze River Delta, China.

Monoaromatic hydrocarbons (MACHs) are a ubiquitous category of volatile compounds found in various environmental media. Despite their prevalence, systematic studies of MACHs on a large regional scale are still lacking. Herein, a comprehensive investigation of the occurrence, seasonal variations, distribution characteristics, and health risks of MACHs was carried out by analyzing soil samples (372 surface soils and 96 soil columns) from 33 typical industrial parks in the Yangtze River Delta (YRD) region. MACHs were detected in all surface soil samples. BTEXS (benzene, toluene, ethylbenzene, xylene, and styrene) were the five predominant congeners with the highest detection frequencies (90.9 %-100 %), collectively accounting for >78.2 % of the total MACHs content. Higher residual levels of MACHs were observed in winter compared to summer (P < 0.01), with total concentrations of 24 MACHs ranging from 30.9 ng/g to 1536 ng/g (median: 135 ng/g) in winter and 16.3 ng/g to 931 ng/g (median: 87.9 ng/g) in summer. Soils collected from the northeast of Jiangsu Province and the southwest of Anhui Province exhibited relatively higher levels of MACHs. On the basis of principal component analysis, we proposed that industrial emissions and vehicle exhaust may be the main sources of MACHs contamination in the soils of YRD industrial parks. Vertically, the concentrations of total MACHs decreased with the soil depth. Soil organic matter (OM) content and the concentration of MACHs in the surface soil layer (0-15 cm) were significant factors influencing the vertical migration and distribution of MACHs (P < 0.05). It was verified that residual MACHs in the soils posed lower lifetime non-carcinogenic and carcinogenic risks to the inhabitants of the study area. The field study provides valuable evidence for the formulation of MACHs pollution control policies in the YRD region.

Unique Tubular BiOBr/g-C3 N4 Heterojunction with Efficient Separation of Charge Carriers for Photocatalytic Nitrogen Fixation.

The industrial ammonia synthesis process consumes a lot of energy and causes serious environmental pollution. As a sustainable approach for ammonia synthesis, photocatalytic nitrogen reduction employing water as the reducing agent has a lot of potential. A simple surfactant-assisted solvothermal method is used to synthesize g-C3 N4 nanotubes with flower-like spherical BiOBr grown inside and outside (BiOBr/g-C3 N4 , BC). The hollow tubular structure realizes the full use of visible light by the multi-scattering effect of light. Large surface areas and more active sites for N2 adsorption and activation are present in the distinctive spatially dispersed hierarchical structures. Particularly, the quick separation and transfer of electrons and holes are facilitated by the sandwich tubular heterojunctions and tight contact interface of BiOBr and g-C3 N4 . The maximal NH3 generation rate of the BiOBr/g-C3 N4 composite catalysts can reach 255.04 µmol⋅ g-1 ⋅ h-1 , and it is 13.9 and 5.8 times that of pure BiOBr and g-C3 N4 . This work provides a novel method for designing and constructing unique heterojunctions for efficient photocatalytic nitrogen fixation.

Constructing NCuS Interface Chemical Bonds over SnS2 for Efficient Solar-Driven Photoelectrochemical Water Splitting.

The restricted charge transfer and slow oxygen evolution reaction (OER) dynamics tremendously hamper the realistic implementation of SnS2 photoanodes for photoelectrochemical (PEC) water splitting. Here, a novel strategy is developed to construct interfacial NCuS bonds between NC skeletons and SnS2 (CuNC@SnS2 ) for efficient PEC water splitting. Compared with SnS2 , the PEC activity of CuNC@SnS2 photoelectrode is tremendously heightened, obtaining a current density of 3.40 mA cm2 at 1.23 VRHE with a negatively shifted onset potential of 0.04 VRHE , which is 6.54 times higher than that of SnS2 . The detailed experimental characterizations and theoretical calculation demonstrate that the interfacial NCuS bonds enhance the OER kinetic, reduce the surface overpotential, facilitate the separation of photon-generated carriers, and provide a fast transmission channel for electrons. This work presents a new approach for modulating charge transfer by interfacial bond design in heterojunction photoelectrodes toward promoting PEC performance and solar energy application.

Membrane tethering of CreER decreases uninduced cell labeling and cytotoxicity while maintaining recombination efficiency.

Genetic lineage tracing is indispensable to unraveling the origin, fate, and plasticity of cells. However, the intrinsic leakiness in the CreER-loxP system raises concerns on data interpretation. Here, we reported the generation of a novel dual inducible CreER-loxP system with superior labeling characteristics. This two-component system consists of membrane localized CreER (mCreER: CD8α-FRB-CS-CreER) and TEV protease (mTEVp: CD8α-FKBP-TEVp), which are fusion proteins incorporated with the chemically induced dimerization machinery. Rapamycin and tamoxifen induce sequential dimerization of FKBP and FRB, cleavage of CreER from the membrane, and translocation into the nucleus. The labeling leakiness in Ad293 cells reduced dramatically from more than 70% to less than 5%. This tight labeling feature depends largely on the association of mCreER with HSP90, which conceals the TEV protease cutting site between FRB and CreER and thus preventing uninduced cleavage of the membrane-tethering CreER. Membrane-bound CreER also diminished significantly cytotoxicity. Our studies showed mCreER under the control of the rat insulin promoter increased labeling specificity in MIN6 islet beta-cells. Viability and insulin secretion of MIN6 cells remained intact. Our results demonstrate that this novel system can provide more stringent temporal and spatial control of gene expression and will be useful in cell fate probing.

Spatial control of in vivo CRISPR-Cas9 genome editing via nanomagnets.

The potential of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9)-based therapeutic genome editing is hampered by difficulties in the control of the in vivo activity of CRISPR-Cas9. To minimize any genotoxicity, precise activation of CRISPR-Cas9 in the target tissue is desirable. Here, we show that, by complexing magnetic nanoparticles with recombinant baculoviral vectors (MNP-BVs), CRISPR-Cas9-mediated genome editing can be activated locally in vivo via a magnetic field. The baculoviral vector was chosen for in vivo gene delivery because of its large loading capacity and ability to locally overcome systemic inactivation by the complement system. We demonstrate that a locally applied magnetic field can enhance the cellular entry of MNP-BVs, thereby avoiding baculoviral vector inactivation and causing a transient transgene expression in the target tissue. Because baculoviral vectors are inactivated elsewhere, gene delivery and in vivo genome editing via MNP-BVs are tissue specific.

Magnetic Iron Oxide Nanoparticles for Disease Detection and Therapy.

Magnetic iron oxide nanoparticles (MIONs) are among the first generation of nanomaterials that have advanced to clinic use. A broad range of biomedical techniques has been developed by combining the versatile nanomagnetism of MIONs with various forms of applied magnetic fields. MIONs can generate imaging contrast and provide mechanical/thermal energy in vivo in response to an external magnetic field, a special feature that distinguishes MIONs from other nanomaterials. These properties offer unique opportunities for nanomaterials engineering in biomedical research and clinical interventions. The past few decades have witnessed the evolution of the applications of MIONs from conventional drug delivery and hyperthermia to the regulation of molecular and cellular processes in the body. Here we review the most recent development in this field, including clinical studies of MIONs and the emerging techniques that may contribute to future innovation in medicine.

Design and Validation of CRISPR/Cas9 Systems for Targeted Gene Modification in Induced Pluripotent Stem Cells.

The CRISPR/Cas9 system is a powerful tool for precision genome editing. The ability to accurately modify genomic DNA in situ with single nucleotide precision opens up new possibilities for not only basic research but also biotechnology applications and clinical translation. In this chapter, we outline the procedures for design, screening, and validation of CRISPR/Cas9 systems for targeted modification of coding sequences in the human genome and how to perform genome editing in induced pluripotent stem cells with high efficiency and specificity.

[Rapid determination of trace volatile organic compounds in ambient air by portable gas chromatography-mass spectrometry].

OBJECTIVE: Direct determination of 35 trace volatile organic compounds in air by portable gas chromatography mass spectrometry( GC-MS). METHODS: The mixed standard gas of different concentration levels was prepared with high purity nitrogen as diluent gas. The samples were injected into the GC-MS by a hand-held probe. Retention time and characteristic ion were used for qualitative analysis, and the internal standard method was usd for quantitation. RESULTS: Under the established experimental conditions, the 35 poisonous substances were separated and determined well. The relative standard deviation was 3. 2%-15. 1%. The average recovery was 75. 1%-121. 4%( n = 6). CONCLUSION: The portable GC-MS method can be used for qualitative and quantitative detection of volatile organic compounds in ambient air.

[Determination of volatile organic compounds in workplace by portable gas-chromatography].

OBJECTIVE: To establish a portable gas chromatography (GC) method for the on-site rapid determination of epoxyethane, furan, dichloromethane, benzene, toluene in workplace. METHODS: By using dynamic dilutor, the volatile organic compounds (VOCs) standard gas was prepared and drawn to the capillary column of GC (PID) for separation and analysis. Based on the retention time and peak area, the VOCs was identified and quantified. RESULTS: At the conditions of 60 degrees C column temperature and 9psi pre-pressure, The five VOCs were well separated. Good linear ranges were obtained within the ranges of 0.45 - 90.72 mg/m3 for epoxyethane, 0.22 - 44.50 mg/m3 for furan, 2.19 - 437.4 mg/m3 for dichloromethane, 0.32 - 13.29 mg/m3 for benzene, 0.38 - 15.68 mg/m3 for toluene, respectively. The correlation coefficient was not less than 0.999. The detection limits were 0.03, 0.04, 0.2, 0.008 and 0.03 mg/m3, respectively. The RSD were 0.79% - 2.4%, 1.5% - 1.9%, 0.97% - 1.8%, 2.3% - 3.2% and 3.4% - 4.6%, respectively. The average recovery were 101.4% - 108.4% 85.49% - 94.98%, 97.78% - 106.2%, 99.29% - 103.5% and 95.54% - 101.7% respectively. By using the 7890A GC method, the relative tolerance for the same gas samples were 0.091% - 8.8% for epoxyethane, 3.8% - 6.7% for furan, 0.77% - 9.4% for dichloromethane, 0.24% - 1.6% for benzene, 0.31% - 8.0% for toluene. CONCLUSION: The method is portable, accurate, sensitive, rapid, and exhibits good separation and anti-interference ability. This method is suitable for the rapid detection of VOCs in workplace and also provides reference VOC detection method for the occupational health standard.

Using artificial microRNA sponges to achieve microRNA loss-of-function in cancer cells.

Widely observed dysregulation of microRNAs (miRNAs) in human cancer has led to substantial speculation regarding possible functions of these short, non-coding RNAs in cancer development and manipulation of miRNA expression to treat cancer. To achieve miRNA loss-of-function, miRNA sponge technology has been developed to use plasmid or viral vectors for intracellular expression of tandemly arrayed, bulged miRNA binding sites complementary to a miRNA target to saturate its ability to regulate natural mRNAs. A strong viral promoter can be used in miRNA sponge vectors to generate high-level expression of the competitive inhibitor transcripts for either transient or long-term inhibition of miRNA function. Taking the advantage of sharing a common seed sequence by members of a miRNA family, this technology is especially useful in knocking down the expression of a family of miRNAs, providing a powerful means for simultaneous inhibition of multiple miRNAs of interest with a single inhibitor. Knockdown of overexpressed oncogenic miRNAs with the technology can be a rational therapeutic strategy for cancer, whereas inhibition of tumor-suppressive miRNAs by the sponges will be useful in deciphering functions of miRNAs in oncogenesis. Herein, we discuss the design of miRNA sponge expression vectors and the use of the vectors to gain better understanding of miRNA's roles in cancer biology and as an alternative tool for anticancer gene therapy.

Genetic rearrangements of variable di-residue (RVD)-containing repeat arrays in a baculoviral TALEN system.

Virus-derived gene transfer vectors have been successfully employed to express the transcription activator-like effector nucleases (TALENs) in mammalian cells. Since the DNA-binding domains of TALENs consist of the variable di-residue (RVD)-containing tandem repeat modules and virus genome with repeated sequences is susceptible to genetic recombination, we investigated several factors that might affect TALEN cleavage efficiency of baculoviral vectors. Using a TALEN system designed to target the AAVS1 locus, we observed increased sequence instability of the TALE repeat arrays when a higher multiplicity of infection (MOI) of recombinant viruses was used to produce the baculoviral vectors. We also detected more deleterious mutations in the TALE DNA-binding domains when both left and right TALEN arms were placed into a single expression cassette as compared to the viruses containing one arm only. The DNA sequence changes in the domains included deletion, addition, substitution, and DNA strand exchange between the left and right TALEN arms. Based on these observations, we have developed a protocol using a low MOI to produce baculoviral vectors expressing TALEN left and right arms separately. Cotransduction of the viruses produced by this optimal protocol provided an improved TALEN cleavage efficiency and enabled effective site-specific transgene integration in human cells.

Zinc finger nuclease-expressing baculoviral vectors mediate targeted genome integration of reprogramming factor genes to facilitate the generation of human induced pluripotent stem cells.

Integrative gene transfer using retroviruses to express reprogramming factors displays high efficiency in generating induced pluripotent stem cells (iPSCs), but the value of the method is limited because of the concern over mutagenesis associated with random insertion of transgenes. Site-specific integration into a preselected locus by engineered zinc-finger nuclease (ZFN) technology provides a potential way to overcome the problem. Here, we report the successful reprogramming of human fibroblasts into a state of pluripotency by baculoviral transduction-mediated, site-specific integration of OKSM (Oct3/4, Klf4, Sox2, and c-myc) transcription factor genes into the AAVS1 locus in human chromosome 19. Two nonintegrative baculoviral vectors were used for cotransduction, one expressing ZFNs and another as a donor vector encoding the four transcription factors. iPSC colonies were obtained at a high efficiency of 12% (the mean value of eight individual experiments). All characterized iPSC clones carried the transgenic cassette only at the ZFN-specified AAVS1 locus. We further demonstrated that when the donor cassette was flanked by heterospecific loxP sequences, the reprogramming genes in iPSCs could be replaced by another transgene using a baculoviral vector-based Cre recombinase-mediated cassette exchange system, thereby producing iPSCs free of exogenous reprogramming factors. Although the use of nonintegrating methods to generate iPSCs is rapidly becoming a standard approach, methods based on site-specific integration of reprogramming factor genes as reported here hold the potential for efficient generation of genetically amenable iPSCs suitable for future gene therapy applications.

Targeted transgene insertion into the AAVS1 locus driven by baculoviral vector-mediated zinc finger nuclease expression in human-induced pluripotent stem cells.

BACKGROUND: The AAVS1 locus is viewed as a 'safe harbor' for transgene insertion into human genome. In the present study, we report a new method for AAVS1 targeting in human-induced pluripotent stem cells (hiPSCs). METHODS: We have developed two baculoviral transduction systems: one to deliver zinc finger nuclease (ZFN) and a DNA donor template for site-specific gene insertion and another to mediate Cre recombinase-mediated cassette exchange system to replace the inserted transgene with a new transgene. RESULTS: Our ZFN system provided the targeted integration efficiency of a Neo-EGFP cassette of 93.8% in G418-selected, stable hiPSC colonies. Southern blotting analysis of 20 AASV1 targeted colonies revealed no random integration events. Among 24 colonies examined for mono- or biallelic AASV1 targeting, 25% of them were biallelically modified. The selected hiPSCs displayed persistent enhanced green fluorescent protein expression and continued the expression of stem cell pluripotency markers. The hiPSCs maintained the ability to differentiate into three germ lineages in derived embryoid bodies and transgene expression was retained in the differentiated cells. After pre-including the loxP-docking sites into the Neo-EGFP cassette, we demonstrated that a baculovirus-Cre/loxP system could be used to facilitate the replacement of the Neo-EGFP cassette with another transgene cassette at the AAVS1 locus. CONCLUSIONS: Given high targeting efficiency, stability in expression of inserted transgene and flexibility in transgene exchange, the approach reported in the present study holds potential for generating genetically-modified human pluripotent stem cells suitable for developmental biology research, drug development, regenerative medicine and gene therapy.

Baculoviral transduction facilitates TALEN-mediated targeted transgene integration and Cre/LoxP cassette exchange in human-induced pluripotent stem cells.

Safety and reliability of transgene integration in human genome continue to pose challenges for stem cell-based gene therapy. Here, we report a baculovirus-transcription activator-like effector nuclease system for AAVS1 locus-directed homologous recombination in human induced pluripotent stem cells (iPSCs). This viral system, when optimized in human U87 cells, provided a targeted integration efficiency of 95.21% in incorporating a Neo-eGFP cassette and was able to mediate integration of DNA insert up to 13.5 kb. In iPSCs, targeted integration with persistent transgene expression was achieved without compromising genomic stability. The modified iPSCs continued to express stem cell pluripotency markers and maintained the ability to differentiate into three germ lineages in derived embryoid bodies. Using a baculovirus-Cre/LoxP system in the iPSCs, the Neo-eGFP cassette at the AAVS1 locus could be replaced by a Hygro-mCherry cassette, demonstrating the feasibility of cassette exchange. Moreover, as assessed by measuring γ-H2AX expression levels, genome toxicity associated with chromosomal double-strand breaks was not detectable after transduction with moderate doses of baculoviral vectors expressing transcription activator-like effector nucleases. Given high targeted integration efficiency, flexibility in transgene exchange and low genome toxicity, our baculoviral transduction-based approach offers great potential and attractive option for precise genetic manipulation in human pluripotent stem cells.