In vivo evaluation of guide-free Cas9-induced safety risks in a pig model.

The CRISPR/Cas9 system has shown great potential for treating human genetic diseases through gene therapy. However, there are concerns about the safety of this system, specifically related to the use of guide-free Cas9. Previous studies have shown that guide-free Cas9 can induce genomic instability in vitro. However, the in vivo safety risks associated with guide-free Cas9 have not been evaluated, which is necessary for the development of gene therapy in clinical settings. In this study, we used doxycycline-inducible Cas9-expressing pigs to evaluate the safety risks of guide-free Cas9 in vivo. Our findings demonstrated that expression of guide-free Cas9 could induce genomic damages and transcriptome changes in vivo. The severity of the genomic damages and transcriptome changes were correlate with the expression levels of Cas9 protein. Moreover, prolonged expression of Cas9 in pigs led to abnormal phenotypes, including a significant decrease in body weight, which may be attributable to genomic damage-induced nutritional absorption and metabolic dysfunction. Furthermore, we observed an increase in whole-genome and tumor driver gene mutations in pigs with long-term Cas9 expression, raising the risk of tumor occurrence. Our in vivo evaluation of guide-free Cas9 in pigs highlights the necessity of considering and monitoring the detrimental effects of Cas9 alone as genome editing via the CRISPR/Cas9 system is implemented in clinical gene therapy. This research emphasizes the importance of further study and implementation of safety measures to ensure the successful and safe application of the CRISPR/Cas9 system in clinical practice.

Exonuclease editor promotes precision of gene editing in mammalian cells.

BACKGROUND: Many efforts have been made to improve the precision of Cas9-mediated gene editing through increasing knock-in efficiency and decreasing byproducts, which proved to be challenging. RESULTS: Here, we have developed a human exonuclease 1-based genome-editing tool, referred to as exonuclease editor. When compared to Cas9, the exonuclease editor gave rise to increased HDR efficiency, reduced NHEJ repair frequency, and significantly elevated HDR/indel ratio. Robust gene editing precision of exonuclease editor was even superior to the fusion of Cas9 with E1B or DN1S, two previously reported precision-enhancing domains. Notably, exonuclease editor inhibited NHEJ at double strand breaks locally rather than globally, reducing indel frequency without compromising genome integrity. The replacement of Cas9 with single-strand DNA break-creating Cas9 nickase further increased the HDR/indel ratio by 453-fold than the original Cas9. In addition, exonuclease editor resulted in high microhomology-mediated end joining efficiency, allowing accurate and flexible deletion of targeted sequences with extended lengths with the aid of paired sgRNAs. Exonuclease editor was further used for correction of DMD patient-derived induced pluripotent stem cells, where 30.0% of colonies were repaired by HDR versus 11.1% in the control. CONCLUSIONS: Therefore, the exonuclease editor system provides a versatile and safe genome editing tool with high precision and holds promise for therapeutic gene correction.

Evaluation of guide-free Cas9-induced genomic damage and transcriptome changes in pig embryos.

Cas9 protein without sgRNAs can induce genomic damage at the cellular level in vitro. However, whether the detrimental effects occur in embryos after Cas9 treatment remains unknown. Here, using pig embryos as subjects, we observed that Cas9 protein transcribed from injected Cas9 mRNA can persist until at least the blastocyst stage. Cas9 protein alone can induce genome damage in preimplantation embryos, represented by the increased number of phosphorylated histone H2AX foci on the chromatin fiber, which led to apoptosis and decreased cell number of blastocysts. In addition, single-blastocyst RNA sequencing confirmed that Cas9 protein without sgRNAs can cause changes in the blastocyst transcriptome, depressing embryo development signal pathways, such as cell cycle, metabolism, and cellular communication-related signal pathways, while activating apoptosis and necroptosis signal pathways, which together resulted in impaired preimplantation embryonic development. These results indicated that attention should be given to the detrimental effects caused by the Cas9 protein when using CRISPR-Cas9 for germline genome editing, especially for the targeted correction of human pathological mutations using germline gene therapy.

Improved USER cloning for TALE assembly and its application to base editing.

Transcription activator-like effectors (TALEs) have been widely used for genome editing, transcriptional regulation, and locus-specific DNA imaging. However, TALEs are difficult to handle in routine laboratories because of their complexity and the considerable time consumed in TALE construction. Here, we described a simple and rapid TALE assembly method based on uracil-specific excision reagent (USER) cloning. Polymerase chain reaction was amplified with TALE trimer templates and deoxyuridine-containing primers. The products were treated with USER at 37°C for 30 min, followed by the treatment of T4 DNA Ligase at 16°C for 30 min. The TALE trimer unit could be rejoined hierarchically to form complete TALE expression vectors with high efficiency. This method was adopted to construct TALE-deaminases, which were used in combination with Cas9 nickases to generate efficient C-to-T or A-to-G base editing while eliminating predictable DNA off-target effects. This improved USER assembly is a simple, rapid, and laboratory-friendly TALE construction technique that will be valuable for DNA targeting.

Base editors: development and applications in biomedicine.

Base editor (BE) is a gene-editing tool developed by combining the CRISPR/Cas system with an individual deaminase, enabling precise single-base substitution in DNA or RNA without generating a DNA double-strand break (DSB) or requiring donor DNA templates in living cells. Base editors offer more precise and secure genome-editing effects than other conventional artificial nuclease systems, such as CRISPR/Cas9, as the DSB induced by Cas9 will cause severe damage to the genome. Thus, base editors have important applications in the field of biomedicine, including gene function investigation, directed protein evolution, genetic lineage tracing, disease modeling, and gene therapy. Since the development of the two main base editors, cytosine base editors (CBEs) and adenine base editors (ABEs), scientists have developed more than 100 optimized base editors with improved editing efficiency, precision, specificity, targeting scope, and capacity to be delivered in vivo, greatly enhancing their application potential in biomedicine. Here, we review the recent development of base editors, summarize their applications in the biomedical field, and discuss future perspectives and challenges for therapeutic applications.

Doxycycline-dependent Cas9-expressing pig resources for conditional in vivo gene nullification and activation.

BACKGROUND: CRISPR-based toolkits have dramatically increased the ease of genome and epigenome editing. SpCas9 is the most widely used nuclease. However, the difficulty of delivering SpCas9 and inability to modulate its expression in vivo hinder its widespread adoption in large animals. RESULTS: Here, to circumvent these obstacles, a doxycycline-inducible SpCas9-expressing (DIC) pig model was generated by precise knock-in of the binary tetracycline-inducible expression elements into the Rosa26 and Hipp11 loci, respectively. With this pig model, in vivo and/or in vitro genome and epigenome editing could be easily realized. On the basis of the DIC system, a convenient Cas9-based conditional knockout strategy was devised through controlling the expression of rtTA component by tissue-specific promoter, which allows the one-step generation of germline-inherited pigs enabling in vivo spatiotemporal control of gene function under simple chemical induction. To validate the feasibility of in vivo gene mutation with DIC pigs, primary and metastatic pancreatic ductal adenocarcinoma was developed by delivering a single AAV6 vector containing TP53-sgRNA, LKB1-sgRNA, and mutant human KRAS gene into the adult pancreases. CONCLUSIONS: Together, these results suggest that DIC pig resources will provide a powerful tool for conditional in vivo genome and epigenome modification for fundamental and applied research.

Multiplexed base editing through Cas12a variant-mediated cytosine and adenine base editors.

Cas12a can process multiple sgRNAs from a single transcript of CRISPR array, conferring advantages in multiplexed base editing when incorporated into base editor systems, which is extremely helpful given that phenotypes commonly involve multiple genes or single-nucleotide variants. However, multiplexed base editing through Cas12a-derived base editors has been barely reported, mainly due to the compromised efficiencies and restricted protospacer-adjacent motif (PAM) of TTTV for wild-type Cas12a. Here, we develop Cas12a-mediated cytosine base editor (CBE) and adenine base editor (ABE) systems with elevated efficiencies and expanded targeting scope, by combining highly active deaminases with Lachnospiraceae bacterium Cas12a (LbCas12a) variants. We confirm that these CBEs and ABEs can perform efficient C-to-T and A-to-G conversions, respectively, on targets with PAMs of NTTN, TYCN, and TRTN. Notably, multiplexed base editing can be conducted using the developed CBEs and ABEs in somatic cells and embryos. These Cas12a variant-mediated base editors will serve as versatile tools for multiplexed point mutation, which is notably important in genetic improvement, disease modeling, and gene therapy.

Double knock-in pig models with elements of binary Tet-On and phiC31 integrase systems for controllable and switchable gene expression.

Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process. Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline. Previous pig models with tetracycline regulatory elements were generated through random integration. This process often resulted in uncertain expression and unpredictable phenotypes, thus hindering their applications. Here, by precise knock-in of binary Tet-On 3G elements into Rosa26 and Hipp11 locus, respectively, a double knock-in reporter pig model was generated. We characterized excellent properties of this system for controllable transgenic expression both in vitro and in vivo. Two attP sites were arranged to flank the tdTomato to switch reporter gene. Single or multiple gene replacement was efficiently and faithfully achieved in fetal fibroblasts and nuclear transfer embryos. To display the flexible application of this system, we generated a pig strain with Dox-inducing hKRASG12D expression through phiC31 integrase-mediated cassette exchange. After eight months of Dox administration, squamous cell carcinoma developed in the nose, mouth, and scrotum, which indicated this pig strain could serve as an ideal large animal model to study tumorigenesis. Overall, the established pig models with controllable and switchable transgene expression system will provide a facilitating platform for transgenic and biomedical research.

AGBE: a dual deaminase-mediated base editor by fusing CGBE with ABE for creating a saturated mutant population with multiple editing patterns.

Establishing saturated mutagenesis in a specific gene through gene editing is an efficient approach for identifying the relationships between mutations and the corresponding phenotypes. CRISPR/Cas9-based sgRNA library screening often creates indel mutations with multiple nucleotides. Single base editors and dual deaminase-mediated base editors can achieve only one and two types of base substitutions, respectively. A new glycosylase base editor (CGBE) system, in which the uracil glycosylase inhibitor (UGI) is replaced with uracil-DNA glycosylase (UNG), was recently reported to efficiently induce multiple base conversions, including C-to-G, C-to-T and C-to-A. In this study, we fused a CGBE with ABE to develop a new type of dual deaminase-mediated base editing system, the AGBE system, that can simultaneously introduce 4 types of base conversions (C-to-G, C-to-T, C-to-A and A-to-G) as well as indels with a single sgRNA in mammalian cells. AGBEs can be used to establish saturated mutant populations for verification of the functions and consequences of multiple gene mutation patterns, including single-nucleotide variants (SNVs) and indels, through high-throughput screening.

Management of Fused Anterior Teeth: Review and Clinical Report.

The phenomenon of fusion is a type of tooth dysplasia, but few studies have systematically described the treatment of this kind of abnormality. This paper summarizes the treatment methods for fused teeth and classifies the management schemes according to whether the pulp is fused. Then, the treatment for a patient with bilateral anterior tooth fusion is reported. After orthodontic treatment, porcelain veneers were used to restore the normal shape and aesthetic appearance of the anterior teeth.

[A review on the genetic mechanism of chromatin remodeling in children with neurodevelopmental disorders].

Neural development is regulated by both external environment and internal signals, and in addition to transcription factors, epigenetic modifications also play an important role. By focusing on the genetic mechanism of ATP-dependent chromatin remodeling in children with neurodevelopmental disorders, this article elaborates on the effect of four chromatin remodeling complexes on neurogenesis and the development and maturation of neurons and neuroglial cells and introduces the clinical research advances in neurodevelopmental disorders.

CRISPR/Cas9-Mediated Gene Correction in Newborn Rabbits with Hereditary Tyrosinemia Type I.

Patients with hereditary tyrosinemia type I (HT1) present acute and irreversible liver and kidney damage during infancy. CRISPR-Cas9-mediated gene correction during infancy may provide a promising approach to treat patients with HT1. However, all previous studies were performed on adult HT1 rodent models, which cannot authentically recapitulate some symptoms of human patients. The efficacy and safety should be verified in large animals to translate precise gene therapy to clinical practice. Here, we delivered CRISPR-Cas9 and donor templates via adeno-associated virus to newborn HT1 rabbits. The lethal phenotypes could be rescued, and notably, these HT1 rabbits reached adulthood normally without 2-(2-nitro-4-trifluoromethylbenzyol)-1,3 cyclohexanedione administration and even gave birth to offspring. Adeno-associated virus (AAV)-treated HT1 rabbits displayed normal liver and kidney structures and functions. Homology-directed repair-mediated precise gene corrections and non-homologous end joining-mediated out-of-frame to in-frame corrections in the livers were observed with efficiencies of 0.90%-3.71% and 2.39%-6.35%, respectively, which appeared to be sufficient to recover liver function and decrease liver and kidney damage. This study provides useful large-animal preclinical data for rescuing hepatocyte-related monogenetic metabolic disorders with precise gene therapy.

ACBE, a new base editor for simultaneous C-to-T and A-to-G substitutions in mammalian systems.

BACKGROUND: Many favorable traits of crops and livestock and human genetic diseases arise from multiple single nucleotide polymorphisms or multiple point mutations with heterogeneous base substitutions at the same locus. Current cytosine or adenine base editors can only accomplish C-to-T (G-to-A) or A-to-G (T-to-C) substitutions in the windows of target genomic sites of organisms; therefore, there is a need to develop base editors that can simultaneously achieve C-to-T and A-to-G substitutions at the targeting site. RESULTS: In this study, a novel fusion adenine and cytosine base editor (ACBE) was generated by fusing a heterodimer of TadA (ecTadAWT/*) and an activation-induced cytidine deaminase (AID) to the N- and C-terminals of Cas9 nickase (nCas9), respectively. ACBE could simultaneously induce C-to-T and A-to-G base editing at the same target site, which were verified in HEK293-EGFP reporter cell line and 45 endogenous gene loci of HEK293 cells. Moreover, the ACBE could accomplish simultaneous point mutations of C-to-T and A-to-G in primary somatic cells (mouse embryonic fibroblasts and porcine fetal fibroblasts) in an applicable efficiency. Furthermore, the spacer length of sgRNA and the length of linker could influence the dual base editing activity, which provided a direction to optimize the ACBE system. CONCLUSION: The newly developed ACBE would expand base editor toolkits and should promote the generation of animals and the gene therapy of genetic diseases with heterogeneous point mutations.

Effect of Co-Cr coating by EB-PVD on the bonding strength between titanium and porcelain. / 纯钛表面电子束蒸镀钴铬合金涂层对钛瓷结合强度的影响.

OBJECTIVES: To investigate the effect of the deposited Co-Cr coatings on the bonding strength of titanium-porcelain restoration, Co-Cr coatings with different thickness were deposited onto the pure titanium substrate by electron beam physical vapor deposition (EB-PVD). METHODS: Sixty pure titanium specimens (25.0 mm×3.0 mm×0.5 mm) were randomly divided into 5 groups. Four Co-Cr coatings with different thickness, including 20, 40, 60 and 80 nm, were deposited onto the specimens (group A, B, C and D, respectively). Sandblasting treatment was selected as a control group (group E). After porcelain fusion, bonding strengths of titanium-porcelain restoration were measured by three-point bending test according to ISO 9 693. Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) were employed to evaluate the morphology and elemental composition of both Co-Cr coatings and interfaces of the titanium-porcelain restoration. RESULTS: The bonding strengths in the group E and the test groups (groups A, B, C, and D) were (30.23±1.94), (36.11±1.17), (43.33±1.17), (39.95±2.22), (38.85±1.10) MPa, respectively. In contrast to the group E, the Co-Cr coatings significantly improved the bonding strength between titanium and porcelain (P<0.05). Moreover, some cracks and pores were found at the titanium-porcelain interface in the group E, while the titanium-porcelain interface in the group B was tight and compact (P<0.05). Meanwhile, comparing to the group E, more residual porcelains were found on the substrate of group B (P<0.05). CONCLUSIONS: Co-Cr coating with appreciate thickness using EB-PVD could significantly improve bonding strength between titanium and porcelain.

Efficient base editing for multiple genes and loci in pigs using base editors.

Cytosine base editors (CBEs) enable programmable C-to-T conversion without DNA double-stranded breaks and homology-directed repair in a variety of organisms, which exhibit great potential for agricultural and biomedical applications. However, all reported cases only involved C-to-T substitution at a single targeted genomic site. Whether C-to-T substitution is effective in multiple sites/loci has not been verified in large animals. Here, by using pigs, an important animal for agriculture and biomedicine, as the subjective animal, we showed that CBEs could efficiently induce C-to-T conversions at multiple sites/loci with the combination of three genes, including DMD, TYR, and LMNA, or RAG1, RAG2, and IL2RG, simultaneously, at the embryonic and cellular levels. CBEs also could disrupt genes (pol gene of porcine endogenous retrovirus) with dozens of copies by introducing multiple premature stop codons. With the CBEs, pigs carrying single gene or multiple gene point mutations were generated through embryo injection or nuclear transfer approach.

Engineering CRISPR/Cpf1 with tRNA promotes genome editing capability in mammalian systems.

CRISPR/Cpf1 features a number of properties that are distinct from CRISPR/Cas9 and provides an excellent alternative to Cas9 for genome editing. To date, genome engineering by CRISPR/Cpf1 has been reported only in human cells and mouse embryos of mammalian systems and its efficiency is ultimately lower than that of Cas9 proteins from Streptococcus pyogenes. The application of CRISPR/Cpf1 for targeted mutagenesis in other animal models has not been successfully verified. In this study, we designed and optimized a guide RNA (gRNA) transcription system by inserting a transfer RNA precursor (pre-tRNA) sequence downstream of the gRNA for Cpf1, protecting gRNA from immediate digestion by 3'-to-5' exonucleases. Using this new gRNAtRNA system, genome editing, including indels, large fragment deletion and precise point mutation, was induced in mammalian systems, showing significantly higher efficiency than the original Cpf1-gRNA system. With this system, gene-modified rabbits and pigs were generated by embryo injection or somatic cell nuclear transfer (SCNT) with an efficiency comparable to that of the Cas9 gRNA system. These results demonstrated that this refined gRNAtRNA system can boost the targeting capability of CRISPR/Cpf1 toolkits.

XIST Derepression in Active X Chromosome Hinders Pig Somatic Cell Nuclear Transfer.

Pig cloning by somatic cell nuclear transfer (SCNT) remains extremely inefficient, and many cloned embryos undergo abnormal development. Here, by profiling transcriptome expression, we observed dysregulated chromosome-wide gene expression in every chromosome and identified a considerable number of genes that are aberrantly expressed in the abnormal cloned embryos. In particular, XIST, a long non-coding RNA gene, showed high ectopic expression in abnormal embryos. We also proved that nullification of the XIST gene in donor cells can normalize aberrant gene expression in cloned embryos and enhance long-term development capacity of the embryos. Furthermore, the increased quality of XIST-deficient embryos was associated with the global H3K9me3 reduction. Injection of H3K9me demethylase Kdm4A into NT embryos could improve the development of pre-implantation stage embryos. However, Kdm4A addition also induced XIST derepression in the active X chromosome and thus was not able to enhance the in vivo long-term developmental capacity of porcine NT embryos.