Donor template delivery by recombinant adeno-associated virus for the production of knock-in mice.

BACKGROUND: The ability of recombinant adeno-associated virus to transduce preimplantation mouse embryos has led to the use of this delivery method for the production of genetically altered knock-in mice via CRISPR-Cas9. The potential exists for this method to simplify the production and extend the types of alleles that can be generated directly in the zygote, obviating the need for manipulations of the mouse genome via the embryonic stem cell route. RESULTS: We present the production data from a total of 13 genetically altered knock-in mouse models generated using CRISPR-Cas9 electroporation of zygotes and delivery of donor repair templates via transduction with recombinant adeno-associated virus. We explore the efficiency of gene targeting at a total of 12 independent genetic loci and explore the effects of allele complexity and introduce strategies for efficient identification of founder animals. In addition, we investigate the reliability of germline transmission of the engineered allele from founder mice generated using this methodology. By comparing our production data against genetically altered knock-in mice generated via gene targeting in embryonic stem cells and their microinjection into blastocysts, we assess the animal cost of the two methods. CONCLUSIONS: Our results confirm that recombinant adeno-associated virus transduction of zygotes provides a robust and effective delivery route for donor templates for the production of knock-in mice, across a range of insertion sizes (0.9-4.7 kb). We find that the animal cost of this method is considerably less than generating knock-in models via embryonic stem cells and thus constitutes a considerable 3Rs reduction.

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.

Genetically engineered birds; pre-CRISPR and CRISPR era†.

Generating biopharmaceuticals in genetically engineered bioreactors continues to reign supreme. Hence, genetically engineered birds have attracted considerable attention from the biopharmaceutical industry. Fairly recent genome engineering methods have made genome manipulation an easy and affordable task. In this review, we first provide a broad overview of the approaches and main impediments ahead of generating efficient and reliable genetically engineered birds, and various factors that affect the fate of a transgene. This section provides an essential background for the rest of the review, in which we discuss and compare different genome manipulation methods in the pre-clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR era in the field of avian genome engineering.

Neuroanatomical organization and functional roles of PVN MC4R pathways in physiological and behavioral regulations.

OBJECTIVE: The paraventricular nucleus of hypothalamus (PVN), an integrative center in the brain, orchestrates a wide range of physiological and behavioral responses. While the PVN melanocortin 4 receptor (MC4R) signaling (PVNMC4R+) is involved in feeding regulation, the neuroanatomical organization of PVNMC4R+ connectivity and its role in other physiological regulations are incompletely understood. Here we aimed to better characterize the input-output organization of PVNMC4R+ neurons and test their physiological functions beyond feeding. METHODS: Using a combination of viral tools, we mapped PVNMC4R+ circuits and tested the effects of chemogenetic activation of PVNMC4R+ neurons on thermoregulation, cardiovascular control, and other behavioral responses beyond feeding. RESULTS: We found that PVNMC4R+ neurons innervate many different brain regions that are known to be important not only for feeding but also for neuroendocrine and autonomic control of thermoregulation and cardiovascular function, including but not limited to the preoptic area, median eminence, parabrachial nucleus, pre-locus coeruleus, nucleus of solitary tract, ventrolateral medulla, and thoracic spinal cord. Contrary to these broad efferent projections, PVNMC4R+ neurons receive monosynaptic inputs mainly from other hypothalamic nuclei (preoptic area, arcuate and dorsomedial hypothalamic nuclei, supraoptic nucleus, and premammillary nucleus), the circumventricular organs (subfornical organ and vascular organ of lamina terminalis), the bed nucleus of stria terminalis, and the parabrachial nucleus. Consistent with their broad efferent projections, chemogenetic activation of PVNMC4R+ neurons not only suppressed feeding but also led to an apparent increase in heart rate, blood pressure, and brown adipose tissue temperature. These physiological changes accompanied acute transient hyperactivity followed by hypoactivity and resting-like behavior. CONCLUSIONS: Our results elucidate the neuroanatomical organization of PVNMC4R+ circuits and shed new light on the roles of PVNMC4R+ pathways in autonomic control of thermoregulation, cardiovascular function, and biphasic behavioral activation.

Generation of auxin inducible degron (AID) knock-in cell lines for targeted protein degradation in mammalian cells.

Targeted protein degradation using degrons, such as the mini-Auxin-inducible degron (mAID), has an advantage over genetic silencing/knockout. However, the efficiency of sgRNA, homologous recombination, tedious expansion, and screening single clones makes the process of tagging endogenous proteins long and laborious. This protocol describes a practical and economical way to obtain AID-tagged endogenous proteins using CRISPR/Cas9-mediated homology-directed repair (HDR). We use the generation of endogenously AID-tagged SPT6 in U2OS cells as an example but provide sufficient details for usage in other cell types. For complete details on the use and execution of this protocol, please refer to Narain et al. (2021).

A protocol for efficient CRISPR-Cas9-mediated knock-in in colorectal cancer patient-derived organoids.

Patient-derived organoids (PDOs) recapitulate the cellular heterogeneity of the original colorectal tumor tissue. Here, we describe a protocol to generate genetically modified PDOs to investigate cancer stem cells. This protocol uses the CRISPR-Cas9 system to knock-in the IRES-EGFP-P2A-iCaspase9 cassette into the 3' UTR of the potential cancer stem cell marker gene, which allows us to investigate their potential for self-replication and pluripotency. We describe the procedure for generating mutant PDOs and their application for stem cell research. For complete details on the generation and use of this protocol, please refer to Okamoto et al. Okamoto et al. (2021).

Alzheimer's disease-causing presenilin-1 mutations have deleterious effects on mitochondrial function.

Mitochondrial dysfunction and oxidative stress are frequently observed in the early stages of Alzheimer's disease (AD). Studies have shown that presenilin-1 (PS1), the catalytic subunit of γ-secretase whose mutation is linked to familial AD (FAD), localizes to the mitochondrial membrane and regulates its homeostasis. Thus, we investigated how five PS1 mutations (A431E, E280A, H163R, M146V, and Δexon9) observed in FAD affect mitochondrial functions. Methods: We used H4 glioblastoma cell lines genetically engineered to inducibly express either the wild-type PS1 or one of the five PS1 mutants in order to examine mitochondrial morphology, dynamics, membrane potential, ATP production, mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), oxidative stress, and bioenergetics. Furthermore, we used brains of PS1M146V knock-in mice, 3xTg-AD mice, and human AD patients in order to investigate the role of PS1 in regulating MAMs formation. Results: Each PS1 mutant exhibited slightly different mitochondrial dysfunction. Δexon9 mutant induced mitochondrial fragmentation while A431E, E280A, H163R, and M146V mutants increased MAMs formation. A431E, E280A, M146V, and Δexon9 mutants also induced mitochondrial ROS production. A431E mutant impaired both complex I and peroxidase activity while M146V mutant only impaired peroxidase activity. All PS1 mutants compromised mitochondrial membrane potential and cellular ATP levels were reduced by A431E, M146V, and Δexon9 mutants. Through comparative profiling of hippocampal gene expression in PS1M146V knock-in mice, we found that PS1M146V upregulates Atlastin 2 (ATL2) expression level, which increases ER-mitochondria contacts. Down-regulation of ATL2 after PS1 mutant induction rescued abnormally elevated ER-mitochondria interactions back to the normal level. Moreover, ATL2 expression levels were significantly elevated in the brains of 3xTg-AD mice and AD patients. Conclusions: Overall, our findings suggest that each of the five FAD-linked PS1 mutations has a deleterious effect on mitochondrial functions in a variety of ways. The adverse effects of PS1 mutations on mitochondria may contribute to MAMs formation and oxidative stress resulting in an accelerated age of disease onset in people harboring mutant PS1.

Knock-in tagging in zebrafish facilitated by insertion into non-coding regions.

Zebrafish provide an excellent model for in vivo cell biology studies because of their amenability to live imaging. Protein visualization in zebrafish has traditionally relied on overexpression of fluorescently tagged proteins from heterologous promoters, making it difficult to recapitulate endogenous expression patterns and protein function. One way to circumvent this problem is to tag the proteins by modifying their endogenous genomic loci. Such an approach is not widely available to zebrafish researchers because of inefficient homologous recombination and the error-prone nature of targeted integration in zebrafish. Here, we report a simple approach for tagging proteins in zebrafish on their N or C termini with fluorescent proteins by inserting PCR-generated donor amplicons into non-coding regions of the corresponding genes. Using this approach, we generated endogenously tagged alleles for several genes that are crucial for epithelial biology and organ development, including the tight junction components ZO-1 and Cldn15la, the trafficking effector Rab11a, the apical polarity protein aPKC and the ECM receptor Integrin ß1b. Our approach facilitates the generation of knock-in lines in zebrafish, opening the way for accurate quantitative imaging studies.

Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes.

CRISPR-Cas9-based genome-editing is a highly efficient and cost-effective method to generate zebrafish loss-of-function alleles. However, introducing patient-specific variants into the zebrafish genome with CRISPR-Cas9 remains challenging. Targeting options can be limited by the predetermined genetic context, and the efficiency of the homology-directed DNA repair pathway is relatively low. Here, we illustrate our efficient approach to develop knock-in zebrafish models using two previously variants associated with hereditary sensory deficits. We employ sgRNA-Cas9 ribonucleoprotein (RNP) complexes that are micro-injected into the first cell of fertilized zebrafish eggs together with an asymmetric, single-stranded DNA template containing the variant of interest. The introduction of knock-in events was confirmed by massive parallel sequencing of genomic DNA extracted from a pool of injected embryos. Simultaneous morpholino-induced blocking of a key component of the non-homologous end joining DNA repair pathway, Ku70, improved the knock-in efficiency for one of the targets. Our use of RNP complexes provides an improved knock-in efficiency as compared to previously published studies. Correct knock-in events were identified in 3-8% of alleles, and 30-45% of injected animals had the target variant in their germline. The detailed technical and procedural insights described here provide a valuable framework for the efficient development of knock-in zebrafish models.

Development of CRISPR-Cas9 knock-in tools for free fatty acid production using the fast-growing cyanobacterial strain Synechococcus elongatus UTEX 2973.

Synechococcus elongatus UTEX 2973 has one of the fastest measured doubling time of cyanobacteria making it an important candidate for metabolic engineering. Traditional genetic engineering methods, which rely on homologous recombination, however, are inefficient, labor-intensive, and time-consuming due to the oligoploidy or polyploidy nature of cyanobacteria and the reliance on unique antibiotic resistance markers. CRISPR-Cas9 has emerged as an effective and versatile editing platform in a wide variety of organisms, but its application for cyanobacterial engineering is limited by the inherent toxicity of Cas9 resulting in poor transformation efficiencies. Here, we demonstrated that a single-plasmid CRISPR-Cas9 system, pCRISPOmyces-2, can effectively knock-in a truncated thioesterase gene from Escherichia coli to generate free fatty acid (FFA) producing mutants of Syn2973. To do so, three parameters were evaluated on the effect of generating recipient colonies after conjugation with pCRISPOmyces-2-based plasmids: 1) a modified conjugation protocol termed streaked conjugation, 2) the deletion of the gene encoding RecJ exonuclease, and 3) single guide RNA (sgRNA) sequence. With the use of the streaked conjugation protocol and a ΔrecJ mutant strain of Syn2973, the conjugation efficiency for the pCRISPomyces-2 plasmid could be improved by 750-fold over the wildtype (WT) for a conjugation efficiency of 2.0 × 10-6 transconjugants/recipient cell. While deletion of the RecJ exonuclease alone increased the conjugation efficiency by 150-fold over the WT, FFA generation was impaired in FFA-producing mutants with the ΔrecJ background, and the large number of poor FFA-producing isolates indicated the potential increase in spontaneous mutation rates. The sgRNA sequence was found to be critical in achieving the desired CRISPR-Cas9-mediated knock-in mutation as the sgRNA impacts conjugation efficiency, likelihood of homogenous recombinants, and free fatty acid production in engineered strains.

Analysis of conventional and alternative CRISPR/Cas9 genome editing to enhance a single-base pair knock-in mutation.

BACKGROUND: The use of CRISPR/Cas9 technologies in generating single-base pair knock-in mutations has recently exploded in the number of methods available. However, with the growing expansion of new technologies, it can be difficult to determine the best method for genome editing. RESULTS: In this study, we evaluated a number of CRISPR/Cas9 approaches for deriving cell lines with knock-in base pair edits to create a phosphorylation mutation and provide a breakdown of editing efficiencies and suggestions for improvement. Overall, our studies suggest that using pre-formed ribonucleoprotein (RNP) complexes is a reliable editing method to generate homozygous single-base pair mutations. We also show that antibiotic selection coupled homologous recombination is an efficient tool for generating highly specific heterozygous mutations. CONCLUSION: The methods and/or combination of methods outlined in this study can be used to help other researchers with similar goals in single-base pair genome editing.

Knock-in models related to Alzheimer's disease: synaptic transmission, plaques and the role of microglia.

BACKGROUND: Microglia are active modulators of Alzheimer's disease but their role in relation to amyloid plaques and synaptic changes due to rising amyloid beta is unclear. We add novel findings concerning these relationships and investigate which of our previously reported results from transgenic mice can be validated in knock-in mice, in which overexpression and other artefacts of transgenic technology are avoided. METHODS: AppNL-F and AppNL-G-F knock-in mice expressing humanised amyloid beta with mutations in App that cause familial Alzheimer's disease were compared to wild type mice throughout life. In vitro approaches were used to understand microglial alterations at the genetic and protein levels and synaptic function and plasticity in CA1 hippocampal neurones, each in relationship to both age and stage of amyloid beta pathology. The contribution of microglia to neuronal function was further investigated by ablating microglia with CSF1R inhibitor PLX5622. RESULTS: Both App knock-in lines showed increased glutamate release probability prior to detection of plaques. Consistent with results in transgenic mice, this persisted throughout life in AppNL-F mice but was not evident in AppNL-G-F with sparse plaques. Unlike transgenic mice, loss of spontaneous excitatory activity only occurred at the latest stages, while no change could be detected in spontaneous inhibitory synaptic transmission or magnitude of long-term potentiation. Also, in contrast to transgenic mice, the microglial response in both App knock-in lines was delayed until a moderate plaque load developed. Surviving PLX5266-depleted microglia tended to be CD68-positive. Partial microglial ablation led to aged but not young wild type animals mimicking the increased glutamate release probability in App knock-ins and exacerbated the App knock-in phenotype. Complete ablation was less effective in altering synaptic function, while neither treatment altered plaque load. CONCLUSIONS: Increased glutamate release probability is similar across knock-in and transgenic mouse models of Alzheimer's disease, likely reflecting acute physiological effects of soluble amyloid beta. Microglia respond later to increased amyloid beta levels by proliferating and upregulating Cd68 and Trem2. Partial depletion of microglia suggests that, in wild type mice, alteration of surviving phagocytic microglia, rather than microglial loss, drives age-dependent effects on glutamate release that become exacerbated in Alzheimer's disease.

A high-fat diet exacerbates the Alzheimer's disease pathology in the hippocampus of the AppNL-F/NL-F knock-in mouse model.

Insulin resistance and diabetes mellitus are major risk factors for Alzheimer's disease (AD), and studies with transgenic mouse models of AD have provided supportive evidence with some controversies. To overcome potential artifacts derived from transgenes, we used a knock-in mouse model, AppNL-F/NL-F , which accumulates Aß plaques from 6 months of age and shows mild cognitive impairment at 18 months of age, without the overproduction of APP. In the present study, 6-month-old male AppNL-F/NL-F and wild-type mice were fed a regular or high-fat diet (HFD) for 12 months. HFD treatment caused obesity and impaired glucose tolerance (i.e., T2DM conditions) in both wild-type and AppNL-F/NL-F mice, but only the latter animals exhibited an impaired cognitive function accompanied by marked increases in both Aß deposition and microgliosis as well as insulin resistance in the hippocampus. Furthermore, HFD-fed AppNL-F/NL-F mice exhibited a significant decrease in volume of the granule cell layer in the dentate gyrus and an increased accumulation of 8-oxoguanine, an oxidized guanine base, in the nuclei of granule cells. Gene expression profiling by microarrays revealed that the populations of the cell types in hippocampus were not significantly different between the two mouse lines, regardless of the diet. In addition, HFD treatment decreased the expression of the Aß binding protein transthyretin (TTR) in AppNL-F/NL-F mice, suggesting that the depletion of TTR underlies the increased Aß deposition in the hippocampus of HFD-fed AppNL-F/NL-F mice.

Generation of Efficient Knock-in Mouse and Human Pluripotent Stem Cells Using CRISPR-Cas9.

A knock-in can generate fluorescent or Cre-reporter under the control of an endogenous promoter. It also generates knock-out or tagged-protein with fluorescent protein and short tags for tracking and purification. Recent advances in genome editing with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) significantly increased the efficiencies of making knock-in cells. Here we describe the detailed protocols of generating knock-in mouse and human pluripotent stem cells (PSCs) by electroporation and lipofection, respectively.

Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells.

The cell biology of circadian clocks is still in its infancy. Here, we describe an efficient strategy for generating knock-in reporter cell lines using CRISPR technology that is particularly useful for genes expressed transiently or at low levels, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins allowing us to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>5 times) compared to PER2 questioning the current model of the circadian oscillator.

Efficient TALEN-mediated gene knockin at the bovine Y chromosome and generation of a sex-reversal bovine.

Functional elucidation of bovine Y-chromosome genes requires available genome editing technologies. Meanwhile, it has yet to be proven whether the bovine Sry gene is the main or single factor involved in the development of the male phenotype in bovine. Here, we efficiently knocked out four Y-linked genes (Sry, ZFY, DDX3Y, and EIF2S3Y) in bovine fetal fibroblasts (BFFs) with transcription activator-like effector nucleases (TALENs) individually. Furthermore, we used TALEN-mediated gene knockin at the Sry gene and generated a sex-reversal bovine by somatic cell nuclear transfer (SCNT). The resulting bovine had only one ovary and was sterile. We demonstrate, for the first time, that the Sry gene is an important sex-determining gene in bovine. Our method lays a solid foundation for detecting the biology of the bovine Y chromosome, as it may provide an alternative biological model system for the study of mammalian sex determination, and new methods for the practical application in agricultural, especially for sex predetermination.

An Optimized Preparation Method for Long ssDNA Donors to Facilitate Quick Knock-In Mouse Generation.

Fluorescent reporter mouse lines and Cre/Flp recombinase driver lines play essential roles in investigating various molecular functions in vivo. Now that applications of the CRISPR/Cas9 genome-editing system to mouse fertilized eggs have drastically accelerated these knock-in mouse generations, the next need is to establish easier, quicker, and cheaper methods for knock-in donor preparation. Here, we reverify and optimize the phospho-PCR method to obtain highly pure long single-stranded DNAs (ssDNAs) suitable for knock-in mouse generation via genome editing. The sophisticated sequential use of two exonucleases, in which double-stranded DNAs (dsDNAs) amplified by a pair of 5'-phosphorylated primer and normal primer are digested by Lambda exonuclease to yield ssDNA and the following Exonuclease III treatment degrades the remaining dsDNAs, enables much easier long ssDNA productions without laborious gel extraction steps. By microinjecting these donor DNAs along with CRISPR/Cas9 components into mouse zygotes, we have effectively generated fluorescent reporter lines and recombinase drivers. To further broaden the applicability, we have prepared long ssDNA donors in higher concentrations and electroporated them into mouse eggs to successfully obtain knock-in embryos. This classical yet improved method, which is regaining attention on the progress of CRISPR/Cas9 development, shall be the first choice for long donor DNA preparation, and the resulting knock-in lines could accelerate life science research.

IL-23 receptor deficiency results in lower bone mass via indirect regulation of bone formation.

The IL-23 receptor (IL-23R) signaling pathway has pleiotropic effects on the differentiation of osteoclasts and osteoblasts, since it can inhibit or stimulate these processes via different pathways. However, the potential role of this pathway in the regulation of bone homeostasis remains elusive. Therefore, we studied the role of IL-23R signaling in physiological bone remodeling using IL-23R deficient mice. Using µCT, we demonstrate that 7-week-old IL-23R-/- mice have similar bone mass as age matched littermate control mice. In contrast, 12-week-old IL-23R-/- mice have significantly lower trabecular and cortical bone mass, shorter femurs and more fragile bones. At the age of 26 weeks, there were no differences in trabecular bone mass and femur length, but most of cortical bone mass parameters remain significantly lower in IL-23R-/- mice. In vitro osteoclast differentiation and resorption capacity of 7- and 12-week-old IL-23R-/- mice are similar to WT. However, serum levels of the bone formation marker, PINP, are significantly lower in 12-week-old IL-23R-/- mice, but similar to WT at 7 and 26 weeks. Interestingly, Il23r gene expression was not detected in in vitro cultured osteoblasts, suggesting an indirect effect of IL-23R. In conclusion, IL-23R deficiency results in temporal and long-term changes in bone growth via regulation of bone formation.

Neuronal ApoE upregulates MHC-I expression to drive selective neurodegeneration in Alzheimer's disease.

Selective neurodegeneration is a critical causal factor in Alzheimer's disease (AD); however, the mechanisms that lead some neurons to perish, whereas others remain resilient, are unknown. We sought potential drivers of this selective vulnerability using single-nucleus RNA sequencing and discovered that ApoE expression level is a substantial driver of neuronal variability. Strikingly, neuronal expression of ApoE-which has a robust genetic linkage to AD-correlated strongly, on a cell-by-cell basis, with immune response pathways in neurons in the brains of wild-type mice, human ApoE knock-in mice and humans with or without AD. Elimination or over-expression of neuronal ApoE revealed a causal relationship among ApoE expression, neuronal MHC-I expression, tau pathology and neurodegeneration. Functional reduction of MHC-I ameliorated tau pathology in ApoE4-expressing primary neurons and in mouse hippocampi expressing pathological tau. These findings suggest a mechanism linking neuronal ApoE expression to MHC-I expression and, subsequently, to tau pathology and selective neurodegeneration.