Comparison and optimization of different CRISPR/Cas9 donor-adapting systems for gene editing.

Gene knock-in in mammalian cells usually uses homology-directed repair (HDR) mechanism to integrate exogenous DNA template into the target genome site. However, HDR efficiency is often low, and the co-localization of exogenous DNA template and target genome site is one of the key limiting factors. To improve the efficiency of HDR mediated by CRISPR/Cas9 system, our team and previous studies fused different adaptor proteins with SpCas9 protein and expressed them. By using their characteristics of binding to specific DNA sequences, many different CRISPR/SpCas9 donor adapter gene editing systems were constructed. In this study, we used them to knock-in eGFP gene at the 3'-end of the terminal exon of GAPDH and ACTB genes in HEK293T cells to facilitate a comparison and optimization of these systems. We utilized an optimized donor DNA template design method, validated the knock-in accuracy via PCR and Sanger sequencing, and assessed the efficiency using flow cytometry. The results showed that the fusion of yGal4BD, hGal4BD, hLacI, hTHAP11 as well as N57 and other adaptor proteins with the C-terminus of SpCas9 protein had no significant effect on its activity. At the GAPDH site, the donor adapter systems of SpCas9 fused with yGal4BD, hGal4BD, hLacI and hTHAP11 significantly improved the knock-in efficiency. At the ACTB site, SpCas9 fused with yGal4BD and hGal4BD significantly improved the knock-in efficiency. Furthermore, increasing the number of BS in the donor DNA template was beneficial to enhance the knock-in efficiency mediated by SpCas9-hTHAP11 system. In conclusion, this study compares and optimizes multiple CRISPR/Cas9 donor adapter gene editing systems, providing valuable insights for future gene editing applications.

CRISPR/Cas9-mediated seamless gene replacement in protoplasts expands the resistance spectrum to TMV-U1 strain in regenerated Nicotiana tabacum.

CRISPR/Cas-based genome editing is now extensively used in plant breeding and continues to evolve. Most CRISPR/Cas current applications in plants focus on gene knock-outs; however, there is a pressing need for new methods to achieve more efficient delivery of CRISPR components and gene knock-ins to improve agronomic traits of crop cultivars. We report here a genome editing system that combines the advantages of protoplast technologies with recent CRISPR/Cas advances to achieve seamless large fragment insertions in the model Solanaceae plant Nicotiana tabacum. With this system, two resistance-related regions of the N' gene were replaced with homologous fragments from the N'alata gene to confer TMV-U1 resistance in the T0 generation of GMO-free plants. Our study establishes a reliable genome-editing tool for efficient gene modifications and provides a detailed description of the optimization process to assist other researchers adapt this system for their needs.

Utilizing flow cytometry sorting signal width to enrich for cells positive to endogenous gene integration of fluorescent proteins.

Endogenous gene knock-in using CRIPSR is becoming the standard for fluorescent tagging of endogenous proteins. Some protocols, particularly those that utilize insert cassettes that carry a fluorescent protein tag, can yield many types of cells with off-target insertions that have diffuse fluorescent signal throughout the whole cell in addition to scarce cells with on-target gene insertions that show the correct sub-cellular localization of the tagged protein. As such, when searching for cells with on-target integration using flow cytometry, the off-target fluorescent cells yield a high percentage of false positives. Here, we show that by changing the gating used to select for fluorescence during flow cytometry sorting, namely utilizing the width of the signal as opposed to the area, we can highly enrich for positively integrated cells. Reproducible gates were created to select even minuscule percentages of correct subcellular signal, and these parameters were validated by fluorescence microscopy. This method is a powerful tool to rapidly enhance the generation of cell lines with correctly integrated gene knock-ins encoding endogenous fluorescent proteins.

An effective double gene knock-in strategy using small-molecule L755507 in the medaka fish (Oryzias latipes).

Homology-directed repair (HDR)-mediated genome editing has become a powerful method for altering chromosomal sequences in a seamless and accurate manner. However, the low efficiency of HDR in most cells hinders the establishment of desired strains harboring accurately modified genomes. To enhance HDR-mediated knock-in events, we explored two approaches, namely low-temperature incubation and chemical compound administration using medaka embryos after microinjection. We validated the performance of each method by calculating the knock-in efficiencies according to the expression area of fluorescent protein in the embryos. The in vivo assay indicated that the reduction in temperature did not promote HDR events, whereas among the nine compounds screened, the small molecule L755507 could enhance the HDR-mediated targeted integration of reporter cassettes. Additionally, the L755507-based approach allowed for the simultaneous integration of two different DNA fragments into the two targeted loci, that is, double knock-in. Our established knock-in system combining L755507, donor plasmids, and the CRISPR/Cas9 nickase system can reduce the workload for genetically modified strain generation, thus accelerating studies on the molecular mechanisms of biological phenomena.

The protective mechanism of Grx2 in ultraviolet-B (UVB)-induced cataract formation.

OBJECTIVE: We established a mouse cataract model by irradiating Grx2 knockout (KO) and knock-in (KI) genetically modified mice with UVB to explore the protective mechanism of Grx2 against UVB lens damage. METHODS: After irradiating Grx2 KO and Grx2 KI mice with UVB lamps, we observed and recorded the general physiological conditions and lens opacity of the mice. The crystalline grading system of the University of Oxford was used to classify the opacity of the lens. Lens reactive oxygen species (ROS) contents were detected using a microplate reader, western blot, and enzyme-linked immunosorbent assay (ELISA) to detect antioxidant and antioxidant enzyme contents. Statistical analysis of the recorded data was performed by using SPSS 19.0 software. RESULTS: After UVB irradiation, the weight of Grx2 KO mice was slightly lower than that of wild-type (WT) mice of the same age. Compared to WT mice, the lens opacity of Grx2 KO mice appeared earlier, the nucleus density of the lens increased, and the opacity increased in the first week after UVB irradiation. Meanwhile, the lenses of Grx2 KI mice remained transparent. The experiment showed that the content of ROS increased, the level of glutathione (GSH) decreased, the content of 8-OHdG increased, and the expression of BCL2 decreased after UVB irradiation. Compared to WT mice, these changes were more significant in Grx2 KO mice. CONCLUSION: This experiment found that knocking out the Grx2 gene accelerated the occurrence and development of UVB-induced cataracts in mice and that Grx2 plays an important role in the oxidative damage caused by UVB radiation by repairing the antioxidant enzymes of the lens. This study provides a new animal model and research ideas for the study of cataract pathogenesis.

Sequence-specific retrotransposition of 28S rDNA-specific LINE R2Ol in human cells.

R2 is a long interspersed element (LINE) found in a specific sequence of the 28S rDNA among a wide variety of animals. Recently, we observed that R2Ol isolated from medaka fish, Oryzias latipes, retrotransposes sequence specifically into the target sequence of zebrafish. Because the 28S target and flanking regions are widely conserved among vertebrates, we examined whether R2Ol can also integrate in a sequence-specific manner in human cells. Using adenovirus-mediated expression of R2Ol constructs, we confirmed an accurate insertion of R2Ol into the 28S target of human 293T cells. However, the R2Ol mutant devoid of endonuclease (EN) activity showed no retrotransposition ability, suggesting that the sequence-specific integration of R2Ol into 28S rDNA occurs via the cleavage activity of EN. By introducing both R2Ol helper virus and donor plasmid in human cells, we succeeded in retrotransposing an exogenous EGFP gene into the 28S target site by the trans-complementation system, which enabled simplification of specific gene knock-in in a time-efficient manner. We believe that R2Ol may provide an alternative targeted gene knock-in method for practical applications such as gene therapy in future.

High-efficiency nonhomologous insertion of a foreign gene into the herpes simplex virus genome.

Efficient, accurate and convenient foreign-gene insertion strategies are crucial for the high-throughput and rapid construction of large DNA viral vectors, but relatively inefficient and labour-intensive methods have limited the application of recombinant viruses. In this study, we applied the nonhomologous insertion (NHI) strategy, which is based on the nonhomologous end joining (NHEJ) repair pathway. Compared to the currently used homologous recombination (HR) strategy, we obtained a higher efficiency of foreign-gene insertion into the herpes simplex virus (HSV) genome that reached 45 % after optimization. By using NHI, we rapidly constructed recombinant reporter viruses using a small amount of clinical viruses, and the recombinant virus was stable for at least ten consecutive passages. The fidelity of NHI ranged from 70-100% and was related to the sequence background of the insertion site according to the sequencing results. Finally, we depict the dynamic process by which the foreign-gene donor plasmid and viral genome are rapidly cleaved by Cas9, as revealed by quantitative pulse analysis. Furthermore, the NHI strategy exerted selection pressure on the wild-type and reverse-integrated viral genomes to efficiently integrate the foreign gene in a predetermined direction. Our results indicate that the use of a rationally designed NHI strategy can allow rapid and efficient foreign gene knock-in into the HSV genome and provide useful guidance for gene insertion into large DNA viral genomes using NHI.

Developing a de novo targeted knock-in method based on in utero electroporation into the mammalian brain.

Genome-editing technology has revolutionized the field of biology. Here, we report a novel de novo gene-targeting method mediated by in utero electroporation into the developing mammalian brain. Electroporation of donor DNA with the CRISPR/Cas9 system vectors successfully leads to knock-in of the donor sequence, such as EGFP, to the target site via the homology-directed repair mechanism. We developed a targeting vector system optimized to prevent anomalous leaky expression of the donor gene from the plasmid, which otherwise often occurs depending on the donor sequence. The knock-in efficiency of the electroporated progenitors reached up to 40% in the early stage and 20% in the late stage of the developing mouse brain. Furthermore, we inserted different fluorescent markers into the target gene in each homologous chromosome, successfully distinguishing homozygous knock-in cells by color. We also applied this de novo gene targeting to the ferret model for the study of complex mammalian brains. Our results demonstrate that this technique is widely applicable for monitoring gene expression, visualizing protein localization, lineage analysis and gene knockout, all at the single-cell level, in developmental tissues.

CRISPR system in the yeast Saccharomyces cerevisiae and its application in the bioproduction of useful chemicals.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) immune systems in bacteria have been used as tools for genome engineering. Thus far, the CRISPR-Cas system has been used in various yeast, bacterial, and mammalian cells. Saccharomyces cerevisiae is a nonpathogenic yeast, classified under "generally recognized as safe", and has long been used to produce consumables such as alcohol or bread. Additionally, recombinant cells of S. cerevisiae have been constructed and used to produce various bio-based chemicals. Some types of CRISPR-Cas system for genetic manipulation have been constructed during the early developmental stages of the CRISPR-Cas system and have been mainly used for gene knock-in and knock-out manipulations. Thereafter, these systems have been used for various novel purposes such as metabolic engineering and tolerance engineering. In this review, we have summarized different aspects of the CRISPR-Cas in the yeast S. cerevisiae, from its basic principles to various applications. This review describes the CRISPR system in S. cerevisiae based on the differences in its origin and efficiency followed by its basic applications; for example, its involvement in gene knock-in and knock-out has been outlined. Finally, advanced applications of the CRISPR system in the bioproduction of useful chemicals have been summarized.

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens.

Beyond its predominant role in human and animal therapy, the CRISPR-Cas9 system has also become an essential tool for plant research and plant breeding. Agronomic applications rely on the mastery of gene inactivation and gene modification. However, if the knock-out of genes by non-homologous end-joining (NHEJ)-mediated repair of the targeted double-strand breaks (DSBs) induced by the CRISPR-Cas9 system is rather well mastered, the knock-in of genes by homology-driven repair or end-joining remains difficult to perform efficiently in higher plants. In this review, we describe the different approaches that can be tested to improve the efficiency of CRISPR-induced gene modification in plants, which include the use of optimal transformation and regeneration protocols, the design of appropriate guide RNAs and donor templates and the choice of nucleases and means of delivery. We also present what can be done to orient DNA repair pathways in the target cells, and we show how the moss Physcomitrella patens can be used as a model plant to better understand what DNA repair mechanisms are involved, and how this knowledge could eventually be used to define more performant strategies of CRISPR-induced gene knock-in.

Malignant hyperthermia, environmental heat stress, and intracellular calcium dysregulation in a mouse model expressing the p.G2435R variant of RYR1.

BACKGROUND: The human p.G2434R variant of the RYR1 gene is most frequently associated with malignant hyperthermia (MH) in the UK. We report the phenotype of a knock-in mouse that expresses the RYR1 variant p.G2435R, which is isogenetic with the human variant. METHODS: We observed the general phenotype; determined the sensitivity of myotubes to caffeine-, KCl, and halothane-induced Ca2+ release; determined the in vivo response to halothane or increased ambient temperature; and determined the in vivo myoplasmic intracellular Ca2+ concentration in skeletal muscle before and during exposure to volatile anaesthetics. RESULTS: RYR1 pG2435R/MH normal (MHS-Heterozygous[Het]) or RYR1 pG2435R/pG2435R (MHS-Homozygous[Hom]) mice were fully viable under typical rearing conditions, although some male MHS-Hom mice died spontaneously. The normalised half-maximal effective concentration (95% confidence interval) for intracellular Ca2+ release in myotubes in response to KCl [MH normal, MHN, 21.4 (19.8-23.1) mM; MHS-Het 16.2 (15.2-17.2) mM; MHS-Hom 11.2 (10.2-12.2) mM] and caffeine (MHN, 5.7 (5-6.3) mM; MHS-Het 4.5 (3.9-5.0) mM; MHS-Hom 1.77 (1.5-2.1) mM] exhibited a gene dose-dependent decrease, and there was a gene dose-dependent increase in halothane sensitivity. Intact animals show a gene dose-dependent susceptibility to MH with volatile anaesthetics or to heat stroke. RYR1 p.G2435R mice had elevated skeletal muscle intracellular resting [Ca2+]i, (values are expressed as mean (SD)) (MHN 123 (3) nM; MHS-Het 156 (16) nM; MHS-Hom 265 (32) nM; P<0.001) and [Na+]i (MHN 8 (0.1) mM; MHS-Het 10 (1) mM; MHS-Hom 14 (0.7) mM; P<0.001) that was further increased by exposure to volatile anaesthetics. CONCLUSIONS: RYR1 pG2435R mice demonstrated gene dose-dependent in vitro and in vivo responses to pharmacological and environmental stressors that parallel those seen in patients with the human RYR1 variant p.G2434R.

FoxO1 Deacetylation Decreases Fatty Acid Oxidation in ß-Cells and Sustains Insulin Secretion in Diabetes.

Pancreatic ß-cell dysfunction contributes to onset and progression of type 2 diabetes. In this state ß-cells become metabolically inflexible, losing the ability to select between carbohydrates and lipids as substrates for mitochondrial oxidation. These changes lead to ß-cell dedifferentiation. We have proposed that FoxO proteins are activated through deacetylation-dependent nuclear translocation to forestall the progression of these abnormalities. However, how deacetylated FoxO exert their actions remains unclear. To address this question, we analyzed islet function in mice homozygous for knock-in alleles encoding deacetylated FoxO1 (6KR). Islets expressing 6KR mutant FoxO1 have enhanced insulin secretion in vivo and ex vivo and decreased fatty acid oxidation ex vivo Remarkably, the gene expression signature associated with FoxO1 deacetylation differs from wild type by only ∼2% of the >4000 genes regulated in response to re-feeding. But this narrow swath includes key genes required for ß-cell identity, lipid metabolism, and mitochondrial fatty acid and solute transport. The data support the notion that deacetylated FoxO1 protects ß-cell function by limiting mitochondrial lipid utilization and raise the possibility that inhibition of fatty acid oxidation in ß-cells is beneficial to diabetes treatment.

Serine 302 Phosphorylation of Mouse Insulin Receptor Substrate 1 (IRS1) Is Dispensable for Normal Insulin Signaling and Feedback Regulation by Hepatic S6 Kinase.

Constitutive activation of the mammalian target of rapamycin complex 1 and S6 kinase (mTORC1→ S6K) attenuates insulin-stimulated Akt activity in certain tumors in part through "feedback" phosphorylation of the upstream insulin receptor substrate 1 (IRS1). However, the significance of this mechanism for regulating insulin sensitivity in normal tissue remains unclear. We investigated the function of Ser-302 in mouse IRS1, the major site of its phosphorylation by S6K in vitro, through genetic knock-in of a serine-to-alanine mutation (A302). Although insulin rapidly stimulated feedback phosphorylation of Ser-302 in mouse liver and muscle, homozygous A302 mice (A/A) and their knock-in controls (S/S) exhibited similar glucose homeostasis and muscle insulin signaling. Furthermore, both A302 and control primary hepatocytes from which Irs2 was deleted showed marked inhibition of insulin-stimulated IRS1 tyrosine phosphorylation and PI3K binding after emetine treatment to raise intracellular amino acids and activate mTORC1 → S6K signaling. To specifically activate mTORC1 in mouse tissue, we deleted hepatic Tsc1 using Cre adenovirus. Although it moderately decreased IRS1/PI3K association and Akt phosphorylation in liver, Tsc1 deletion failed to cause glucose intolerance or promote hyperinsulinemia in mixed background A/A or S/S mice. Moreover, Tsc1 deletion failed to stimulate phospho-Ser-302 or other putative S6K sites within IRS1, whereas ribosomal S6 protein was constitutively phosphorylated. Following acute Tsc1 deletion from hepatocytes, Akt phosphorylation, but not IRS1/PI3K association, was rapidly restored by treatment with the mTORC1 inhibitor rapamycin. Thus, within the hepatic compartment, mTORC1 → S6K signaling regulates Akt largely through IRS-independent means with little effect upon physiologic insulin sensitivity.

The expanding footprint of CRISPR/Cas9 in the plant sciences.

CRISPR/Cas9 has evolved and transformed the field of biology at an unprecedented pace. From the initial purpose of introducing a site specific mutation within a genome of choice, this technology has morphed into enabling a wide array of molecular applications, including site-specific transgene insertion and multiplexing for the simultaneous induction of multiple cleavage events. Efficiency, specificity, and flexibility are key attributes that have solidified CRISPR/Cas9 as the genome-editing tool of choice by scientists from all areas of biology. Within the field of plant biology, several CRISPR/Cas9 technologies, developed in other biological systems, have been successfully implemented to probe plant gene function and to modify specific crop traits. It is anticipated that this trend will persist and lead to the development of new applications and modifications of the CRISPR technology, adding to an ever-expanding collection of genome-editing tools. We envision that these tools will bestow plant researchers with new utilities to alter genome complexity, engineer site-specific integration events, control gene expression, generate transgene-free edited crops, and prevent or cure plant viral disease. The successful implementation of such utilities will represent a new frontier in plant biotechnology.

Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids.

Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.

Study on the Role of ampG in the Regulation of Plasmid-Mediated ampC -Induced Expression in Klebsiella pneumoniae.

Objective: In this study, we constructed ampG knock-out and knock-in strains from a clinically isolated Kp1strain carrying ampR-ampC in its plasmid and compared them with the Kp NTUH-K2044 strain to investigate the relationship between ampG and ampR-ampC-induced expression. Methods: We created the ampG gene deletion mutant strains Kp1-ΔampG and Kp NTUH-K2044-ΔampG with pKO3-km plasmid using homologous recombination technology. We constructed the Kp NTUH-K2044-RC and Kp NTUH-K2044-ΔampG-RC drug resistance model strains with plasmid pACYC184. We constructed the ampG knock-in strains by introducing the ampG genes of Kp1, Enterobacter cloacae 029M, Pseudomonas aeruginosa PAO1, Escherichia coli ATCC25922, and Salmonella typhimurium LT2 into the ampG gene-deleted strains with carrier pet-30a. Real-time polymerase chain reaction (real-time PCR) was used to detect the relative expressions of ampC and ampG mRNAs. Results: Compared with Kp1, the induction phenotype of the ampC of Kp1-ΔampG strain disappeared, the ampC expression was reduced, and the minimal inhibitory concentration (MIC) values of cefoxitin and ceftazidime significant decrease from 128 µg/mL to 1 µg/mL. Based on Kp1, five strain were successfully constructed to complement the ampG genes from five knock-in strain, and all of the above complemented strains showed inducible expression of ampC and restored the expression of ampG to varying degrees, as well as restored resistance to the antimicrobial drugs cefoxitin and ceftazidime (P < 0.05). The ampC and ampG genes were barely expressed in Kp NTUH-K2044-ΔampG-RC when compared with Kp NTUH-K2044-RC. The expressions of ampG and ampC in each knock-in strain were recovered, the induction phenotype of ampC was restored, and the MIC values of cefoxitin and ceftazidime were increased. (P < 0.05). Conclusion: In this study, we found that ampG was an essential regulator for the plasmid-mediated ampC-induced expression in K. pneumoniae.

Efficient Large DNA Fragment Knock-in by Long dsDNA with 3'-Overhangs Mediated CRISPR Knock-in (LOCK) in Mammalian Cells.

An efficient and precise genome-editing approach is in high demand in any molecular biology or cell biology laboratory worldwide. However, despite a recent rapid progress in the toolbox tailored for precise genome-editing, including the base editors and prime editors, there is still a need for a cost-effective knock-in (KI) approach amenable for long donor DNA cargos with high efficiency. By harnessing the high-efficient double-strand break (DSB) repair pathway of microhomology-mediated end joining, we previously showed that a specially designed 3'-overhang double-strand DNA (odsDNA) donor harboring 50-nt homology arm (HA) allows high-efficient exogenous DNA KI when combined with CRISPR-Cas9 technology. The lengths of the 3'-overhangs of odsDNA donors could be manipulated by the five consecutive phosphorothioate (PT) modifications. In this protocol, we detail the stepwise procedures to conduct the LOCK (Long dsDNA with 3'-Overhangs mediated CRISPR Knock-in) method for gene-sized (~1-3 kb) KI in mammalian cells.

P21 Overexpression Promotes Cell Death and Induces Senescence in Human Glioblastoma.

High-grade gliomas are the most common and aggressive adult primary brain tumors with a median survival of only 12-15 months. Current standard therapy consists of maximal safe surgical resection followed by DNA-damaging agents, such as irradiation and chemotherapy that can delay but not prevent inevitable recurrence. Some have interpreted glioma recurrence as evidence of glioma stem cells which persist in a relatively quiescent state after irradiation and chemotherapy, before the ultimate cell cycle re-entry and glioma recurrence. Conversely, latent cancer cells with a therapy-induced senescent phenotype have been shown to escape senescence, giving rise to more aggressive stem-like tumor cells than those present in the original tumor. Therefore, approaches are needed to either eliminate or keep these glioma initiating cells in a senescent state for a longer time to prolong survival. In our current study, we demonstrate that the radiation-induced cell cycle inhibitor P21 can provide a powerful route to induce cell death in short-term explants of PDXs derived from three molecularly diverse human gliomas. Additionally, cells not killed by P21 overexpression were maintained in a stable senescent state for longer than control cells. Collectively, these data suggest that P21 activation may provide an attractive therapeutic target to improve therapeutic outcomes.

Functional replacement of myostatin with GDF-11 in the germline of mice.

BACKGROUND: Myostatin (MSTN) is a transforming growth factor-ß superfamily member that acts as a major regulator of skeletal muscle mass. GDF-11, which is highly related to MSTN, plays multiple roles during embryonic development, including regulating development of the axial skeleton, kidneys, nervous system, and pancreas. As MSTN and GDF-11 share a high degree of amino acid sequence identity, behave virtually identically in cell culture assays, and utilize similar regulatory and signaling components, a critical question is whether their distinct biological functions result from inherent differences in their abilities to interact with specific regulatory and signaling components or whether their distinct biological functions mainly reflect their differing temporal and spatial patterns of expression. METHODS: We generated and characterized mice in which we precisely replaced in the germline the portion of the Mstn gene encoding the mature C-terminal peptide with the corresponding region of Gdf11. RESULTS: In mice homozygous for the knock-in allele, all of the circulating MSTN protein was replaced with GDF-11, resulting in ~ 30-40-fold increased levels of circulating GDF-11. Male mice homozygous for the knock-in allele had slightly decreased muscle weights, slightly increased weight gain in response to a high-fat diet, slightly increased plasma cholesterol and HDL levels, and significantly decreased bone density and bone mass, whereas female mice were mostly unaffected. CONCLUSIONS: GDF-11 appears to be capable of nearly completely functionally replacing MSTN in the control of muscle mass. The developmental and physiological consequences of replacing MSTN with GDF-11 are strikingly limited.

From DNA break repair pathways to CRISPR/Cas-mediated gene knock-in methods.

Various DNA breaks created via programmable CRISPR/Cas9 nuclease activity results in different intracellular DNA break repair pathways. Based on the cellular repair pathways, CRISPR-based gene knock-in methods can be categorized into two major strategies: 1) Homology-independent strategies which are targeted insertion events based on non-homologous end joining, and 2) Homology-dependent strategies which are targeted insertion events based on the homology-directed repair. This review elaborates on various gene knock-in methods in mammalian cells using the CRISPR/Cas9 system and in sync with DNA-break repair pathways. Gene knock-in methods are applied in functional genomics and gene therapy. To compensate or correct genetic defects, different CRISPR-based gene knock-in strategies can be used. Thus, researchers need to make a conscious decision about the most suitable knock-in method. For a successful gene-targeted insertion, some determinant factors should be considered like cell cycle, dominant DNA repair pathway, size of insertions, and donor properties. In this review, different aspects of each gene knock-in strategy are discussed to provide a framework for choosing the most appropriate gene knock-in method in different applications.