Analysis of Brain, Blood, and Testis Phenotypes Lacking the Vps13a Gene in C57BL/6N Mice.

The Vps13a gene encodes a lipid transfer protein called VPS13A, or chorein, associated with mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), mitochondria-endosomes, and lipid droplets. This protein plays a crucial role in inter-organelle communication and lipid transport. Mutations in the VPS13A gene are implicated in the pathogenesis of chorea-acanthocytosis (ChAc), a rare autosomal recessive neurodegenerative disorder characterized by chorea, orofacial dyskinesias, hyperkinetic movements, seizures, cognitive impairment, and acanthocytosis. Previous mouse models of ChAc have shown variable disease phenotypes depending on the genetic background. In this study, we report the generation of a Vps13a flox allele in a pure C57BL/6N mouse background and the subsequent creation of Vps13a knockout (KO) mice via Cre-recombination. Our Vps13a KO mice exhibited increased reticulocytes but not acanthocytes in peripheral blood smears. Additionally, there were no significant differences in the GFAP- and Iba1-positive cells in the striatum, the basal ganglia of the central nervous system. Interestingly, we observed abnormal spermatogenesis leading to male infertility. These findings indicate that Vps13a KO mice are valuable models for studying male infertility and some hematological aspects of ChAc.

CRISPR-mediated knock-in in the mouse embryo using long single stranded DNA donors synthesised by biotinylated PCR.

Successful gene knock-in by CRISPR-Cas9 in the mouse zygote requires three components; guideRNA, Cas9 protein and a suitable donor template, which usually comprises homology flanked insert sequence. Recently, long single stranded DNA (lssDNA) donors have emerged as a popular choice of DNA donor, outperforming dsDNA templates in terms of knock-in efficiency for gene tagging and generating conditional alleles. The generation of these donors can be achieved through several methods that may introduce errors in the sequence, result in poor yields, and contain dsDNA contamination. We have developed our own cost-effective lssDNA synthesis methodology that results in high purity, sequence verified, low contamination lssDNA donors. We provide a detailed methodology on the design and generation of such donors for gene tagging experiments and generating conditional alleles.

Multifunctional Alleles: A novel method for the generation of "All-In-One" null and conditional alleles.

The engineering of conditional alleles has evolved from simple floxing of regions of genes to more elaborate methods. Previously, we developed Conditional by Inversion (COIN), an allele design that utilizes an exon-splitting intron and an invertible genetrap-like module (COIN module) to create null alleles upon Cre-mediated inversion. Here we build upon COINs by generating a new Multifunctional Allele (MFA), that utilizes a single gene-targeting step and three site-specific recombination systems, to generate four allelic states: 1. The initial MFA (generated upon targeting) functions as a null with reporter (plus drug selection cassette) allele, wherein the gene of interest is inactivated by both inversion of a critical region of its coding sequence and simultaneous insertion of a reporter gene. MFAs can also be used as 'reverse-conditional' alleles as they are functionally wild type when they are converted to COIN alleles. 2. Null with reporter (minus drug selection cassette), wherein the selection cassette, the inverted critical region, and the COIN module are removed. 3. COIN-based conditional-null via removal of the selection cassette and reporter and simultaneous re-inversion of the critical region of the target. 4. Inverted COIN allele, wherein the COIN allele in turn is reconverted to a null allele by taking advantage of the COIN module's gene trap while simultaneously deleting the critical region.

Using CRISPR/Cas9 engineering to generate a mouse with a conditional knockout allele for the promyelocytic leukemia zinc finger transcription factor.

The promyelocytic leukemia zinc finger (PLZF) transcription factor mediates a wide-range of biological processes. Accordingly, perturbation of PLZF function results in a myriad of physiologic defects, the most conspicuous of which is abnormal skeletal patterning. Although whole body knockout of Plzf in the mouse (Plzf KO ) has significantly expanded our understanding of Plzf function in vivo, a conditional knockout mouse model that enables tissue or cell-type specific ablation of Plzf has not been developed. Therefore, we used CRISPR/Cas 9 gene editing to generate a mouse model in which exon 2 of the murine Plzf gene is specifically flanked (or floxed) by LoxP sites (Plzf f/f ). Crossing our Plzf f/f mouse with a global cre-driver mouse to generate the Plzf d/d bigenic mouse, we demonstrate that exon 2 of the Plzf gene is ablated in the Plzf d/d bigenic. Similar to the previously reported Plzf KO mouse, the Plzf d/d mouse exhibits a severe defect in skeletal patterning of the hindlimb, indicating that the Plzf f/f mouse functions as designed. Therefore, studies in this short technical report demonstrate that the Plzf f/f mouse will be useful to investigators who wish to explore the role of the Plzf transcription factor in a specific tissue or cell-type.

Deletion of exons encoding carboxypeptidase domain of Nna1 results in Purkinje cell degeneration (pcd) phenotype.

Purkinje cell degeneration (pcd) was first identified in a spontaneous mouse mutant showing cerebellar ataxia. In addition to cerebellar Purkinje cells (PCs), retinal photoreceptors, mitral cells in the olfactory bulb, and a discrete subpopulation of thalamic neurons also degenerate in the mutant brains. The gene responsible for the pcd mutant is Nna1, also known as ATP/GTP binding protein 1 or cytosolic carboxypeptidase-like 1, which encodes a zinc carboxypeptidase protein. To investigate pathogenesis of the pcd mutation in detail, we generated a conditional Nna1 allele targeting the carboxypeptidase domain at C-terminus. After Cre recombination and heterozygous crossing, we generated Nna1 knockout (KO) mice and found that the Nna1 KO mice began to show cerebellar ataxia at postnatal day 20 (P20). Most PCs degenerated until 4-week-old, except lobule X. Activated microglia and astrocytes were also observed in the Nna1 KO cerebellum. In the mutant brain, the Nna1 mRNA level was dramatically reduced, suggesting that nonsense-mediated mRNA decay occurs in it. Since the Nna1 protein acts as a de-glutamatase on the C-terminus of α-tubulin and ß-tubulin, increased polyglutamylated tubulin was detected in the Nna1 KO cerebellum. In addition, the endoplasmic reticulum stress marker, C/EBP homologous protein (CHOP), was up-regulated in the mutant PCs. We report the generation of a functional Nna1 conditional allele and possible mechanisms of PC death in the Nna1 KO in the cerebellum. OPEN PRACTICES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Generation and characterization of a conditional allele of Interferon Regulatory Factor 6.

Interferon Regulatory Factor 6 (IRF6) is a critical regulator of differentiation, proliferation, and migration of keratinocytes. Mutations in IRF6 cause two autosomal dominant disorders characterized by cleft lip with or without cleft palate. In addition, DNA variation in IRF6 confers significant risk for non-syndromic cleft lip and palate. IRF6 is also implicated in adult onset development and disease processes, including mammary gland development and squamous cell carcinoma. Mice homozygous for a null allele of Irf6 die shortly after birth due to severe skin, limb, and craniofacial defects, thus impeding the study of gene function after birth. To circumvent this, a conditional allele of Irf6 was generated. To validate the functionality of the conditional allele, we used three "deleter" Cre strains: Gdf9-Cre, CAG-Cre, and Ella-Cre. When Cre expression was driven by the Gdf9-Cre or CAG-Cre transgenes, 100% recombination was observed as indicated by DNA genotyping and phenotyping. In contrast, use of the Ella-Cre transgenic line resulted in incomplete recombination, despite expression at the one-cell stage. In sum, we generated a novel tool to delete Irf6 in a tissue specific fashion, allowing for study of gene function past perinatal stages. However, recombination efficiency of this allele was dictated by the Cre-driver used.

Generation of a Magoh conditional allele in mice.

Magoh encodes a core component of the exon junction complex (EJC), which binds mRNA and regulates mRNA metabolism. Magoh is highly expressed in proliferative tissues during development. EJC components have been implicated in several developmental disorders including TAR syndrome, Richieri-Costa-Pereira syndrome, and intellectual disability. Existing germline null Magoh mice are embryonic lethal as homozygotes and perinatal lethal as heterozygotes, precluding detailed analysis of embryonic and postnatal functions. Here, we report the generation of a new genetic tool to dissect temporal and tissue-specific roles for Magoh in development and adult homeostasis. This Magoh conditional allele has two loxP sites flanking the second exon. Ubiquitous Cre-mediated deletion of the floxed allele in a heterozygous mouse (Magoh(del/+) ) causes 50% reduction of both Magoh mRNA and protein. Magoh(del/+) mice exhibit both microcephaly and hypopigmentation, thus phenocopying germline haploinsufficient Magoh mice. Using Emx1-Cre, we further show that conditional Magoh deletion in neural progenitors during embryonic development also causes microcephaly. We anticipate this novel conditional allele will be a valuable tool for assessing tissue-specific roles for Magoh in mammalian development and postnatal processes.

Genetic Analysis of the ts-Lethal Mutant Δpa0665/pTS-pa0665 Reveals Its Role in Cell Morphology and Oxidative Phosphorylation in Pseudomonas aeruginosa.

Pa0665 in Pseudomonas aeruginosa shares homologous sequences with that of the essential A-type iron-sulfur (Fe-S) cluster insertion protein ErpA in Escherichia coli. However, its essentiality in P. aeruginosa and its complementation with E. coli erpA has not been experimentally examined. To fulfill this task, we constructed plasmid-based ts-mutant Δpa0665/pTS-pa0665 using a three-step protocol. The mutant displayed growth defects at 42 °C, which were complemented by expressing ec.erpA. Microscopic observations indicated a petite cell phenotype for Δpa0665/pTS-pa0665 at 42 °C, correlated with the downregulation of the oprG gene. RNA sequencing revealed significant transcriptional changes in genes associated with the oxidative phosphorylation (OXPHOS) system, aligning with reduced ATP levels in Δpa0665/pTS-pa0665 under 42 °C. Additionally, the ts-mutant showed heightened sensitivity to H2O2 at 42 °C. Overall, our study demonstrates the essential role of pa0665 for OXPHOS function and is complemented by ec.erpA. We propose that the plasmid-based ts-allele is useful for genetic analysis of essential genes of interest in P. aeruginosa.

Genetic Analysis of the Plasmid-Based Temperature-Lethal Mutant pa1792|lpxH(Ts) in Pseudomonas aeruginosa.

Many enzymes in the Raetz pathway for lipid A biosynthesis in Escherichia coli are essential. A homologous protein Pa1792|LpxH in Pseudomonas aeruginosa is known to complement the loss of LpxH in E. coli. Genome-wide transposon-insertion sequencing analysis indicates that lpxH is essential in P. aeruginosa. However, genetic analysis of lpxH in P. aeruginosa has not been carried out, partly because the conditional alleles of essential genes are not readily constructed. In this study, we first constructed a plasmid-based temperature-sensitive mutant ΔlpxH/pTS-lpxH or lpxH(Ts) in P. aeruginosa PAO1. Spot-plating assay indicated that lpxH(Ts) was lethal at a restrictive temperature, confirming its essentiality for growth. Microscopic analysis revealed that lpxH(Ts) exhibited an oval-shaped morphology, suggesting that lpxH was required for rod-shape formation. SDS-PAGE and Western blotting analysis showed that lpxH(Ts) failed to synthesize lipid A, consistent with its function in lipid A biosynthesis. Strong expression of lpxH but not the non-homologous isoenzyme lpxI or lpxG impeded growth and caused cell lysis, implying that lpxH-specific cofactors were required for this toxic effect in P. aeruginosa. Together, our results demonstrate that lpxH is essential for lipid A biosynthesis, rod-shaped growth, and viability in P. aeruginosa. We propose that this plasmid-based conditional allele is a useful tool for the genetic study of essential genes in P. aeruginosa.

Analysis of the Plasmid-Based ts-Mutant ΔfabA/pTS-fabA Reveals Its Lethality under Aerobic Growth Conditions That Is Suppressed by Mild Overexpression of desA at a Restrictive Temperature in Pseudomonas aeruginosa.

It is uncertain whether PA1610|fabA is essential or dispensable for growth on LB-agar plates under aerobic conditions in Pseudomonas aeruginosa PAO1. To examine its essentiality, we disrupted fabA in the presence of a native promoter-controlled complementary copy on ts-plasmid. In this analysis, we showed that the plasmid-based ts-mutant ΔfabA/pTS-fabA failed to grow at a restrictive temperature, consistent with the observation by Hoang and Schweizer (T. T. Hoang, H. P. Schweizer, J Bacteriol 179:5326-5332, 1997, https://doi.org/10.1128/jb.179.17.5326-5332.1997), and expanded on this by showing that ΔfabA exhibited curved cell morphology. On the other hand, strong induction of fabA-OE or PA3645|fabZ-OE impeded the growth of cells displaying oval morphology. Suppressor analysis revealed a mutant sup gene that suppressed a growth defect but not cell morphology of ΔfabA. Genome resequencing and transcriptomic profiling of sup identified PA0286|desA, whose promoter carried a single-nucleotide polymorphism (SNP), and transcription was significantly upregulated (level increase of >2-fold, P < 0.05). By integration of the SNP-bearing promoter-controlled desA gene into the chromosome of ΔfabA/pTS-fabA, we showed that the SNP is sufficient for ΔfabA to phenocopy the sup mutant. Furthermore, mild induction of the araC-PBAD-controlled desA gene but not desB rescued ΔfabA. These results validated that mild overexpression of desA fully suppressed the lethality but not the curved cell morphology of ΔfabA. Similarly, Zhu et al. (Zhu K, Choi K-H, Schweizer HP, Rock CO, Zhang Y-M, Mol Microbiol 60:260-273, 2006, https://doi.org/10.1111/j.1365-2958.2006.05088.x) showed that multicopy desA partially alleviated the slow growth phenotype of ΔfabA, the difference in which was that ΔfabA was viable. Taken together, our results demonstrate that fabA is essential for aerobic growth. We propose that the plasmid-based ts-allele is useful for exploring the genetic suppression interaction of essential genes of interest in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen whose multidrug resistance demands new drug development. Fatty acids are essential for viability, and essential genes are ideal drug targets. However, the growth defect of essential gene mutants can be suppressed. Suppressors tend to be accumulated during the construction of essential gene deletion mutants, hampering the genetic analysis. To circumvent this issue, we constructed a deletion allele of fabA in the presence of a native promoter-controlled complementary copy in the ts-plasmid. In this analysis, we showed that ΔfabA/pTS-fabA failed to grow at a restrictive temperature, supporting its essentiality. Suppressor analysis revealed desA, whose promoter carried a SNP and whose transcription was upregulated. We validated that both the SNP-bearing promoter-controlled and regulable PBAD promoter-controlled desA suppressed the lethality of ΔfabA. Together, our results demonstrate that fabA is essential for aerobic growth. We propose that plasmid-based ts-alleles are suitable for genetic analysis of essential genes of interest.

One-step generation of a conditional allele in mice using a short artificial intron.

Despite tremendous advances in genome editing technologies, generation of conditional alleles in mice has remained challenging. Recent studies in cells have successfully made use of short artificial introns to engineer conditional alleles. The approach consists of inserting a small cassette within an exon of a gene using CRISPR-Cas9 technology. The cassette, referred to as Artificial Intron version 4 (AIv4), contains sequences encoding a splice donor, essential intronic sequences flanked by loxP sites and a splice acceptor site. Under normal conditions, the artificial intron is removed by the splicing machinery, allowing for proper expression of the gene product. Following Cre-mediated recombination of the two loxP sites, the intron is disabled, and splicing can no longer occur. The remaining intronic sequences create a frameshift and early translation termination. Here we describe the application of this technology to engineer a conditional allele in mice using Scyl1 as a model gene. Insertion of the cassette occurred in 17% of edited mice obtained from pronuclear stage zygote microinjection. Mice homozygous for the insertion expressed SCYL1 at levels comparable to wild-type mice and showed no overt abnormalities associated with the loss of Scyl1 function, indicating the proper removal of the artificial intron. Inactivation of the cassette via Cre-mediated recombination in vivo occurred at high frequency, abrogated SCYL1 protein expression, and resulted in loss-of-function phenotypes. Our results broaden the applicability of this approach to engineering conditional alleles in mice.

Generation of an Allelic Series at the Ahr Locus Using an Edited Recombinant Approach.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and a member of the PER-ARNT-SIM (PAS) superfamily of environmental sensors. The AHR is involved in a series of biological processes including adaptive metabolism of xenobiotics, toxicity of certain environmental pollutants, vascular development, fertility, and immune function. Mouse models, including the Ahr null and Ahr conditional null (Ahrfx) mice, are widely used for the study of AHR-mediated biology and toxicity. The Ahr conditional null mouse harbors the low-affinity Ahrd allele that exhibits approximately a 10-fold lower binding affinity for certain xenobiotic AHR ligands than the widely used C57BL/6 mouse that harbors the higher affinity Ahrb1 allele. Here, we report a novel mouse model that introduces a V375A polymorphism that converts the low-affinity allele into a high-affinity allele, offering a more sensitive conditional model. In the generation of this novel conditional allele, two additional mutants arose, including a 3-bp deletion in the PAS-B domain (AhrNG367R) and an early termination codon in the PAS-B domain (AhrTer383). The AhrNG367R allele presents as a phenocopy of the null and the AhrTer383 allele presents as an antimorph when assessing for the ductus venosus and liver lobe weight endpoints. These new models represent a series of tools that will be useful in further characterizing AHR biology.

Efficient Generation of Large-Fragment Knock-In Mouse Models Using 2-Cell (2C)-Homologous Recombination (HR)-CRISPR.

Generating large-fragment knock-ins, such as reporters, conditional alleles, or humanized alleles, directly in mouse embryos is still a challenging feat. We have developed 2C-HR-CRISPR, a technology that allows highly efficient (10-50%) and rapid (generating founders in 2 months) targeting of large DNA fragments. Key to this strategy is the delivery of CRISPR reagents into 2-cell-stage mouse embryos, taking advantage of the high homologous recombination activity during the long G2 cell cycle phase at this stage. Furthermore, by exploiting a Cas9-monomeric streptavidin (Cas-mSA) and biotinylated PCR template (BioPCR) system to localize the repair template to specific double strand breaks, the efficiency can be further improved to up to 95%. Here we provide a procedure to generate large-fragment knock-in mouse models using 2C-HR-CRISPR. We first describe the principles for designing single guide RNAs and repair templates but refer to published manuscripts and protocols for molecular cloning methods or commercial sources for these reagents. We then describe two unique aspects of 2C-HR-CRISPR that are critical for success: (1) production of the CRISPR reagents for 2C-HR-CRISPR, particularly for applying the Cas9-mSA/BioPCR method, and (2) microinjection of mouse embryos at the 2-cell stage. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Single guide RNA and repair template design Basic Protocol 2: Preparing reagents for 2C-HR-CRISPR Basic Protocol 3: Microinjecting 2-cell-stage mouse embryos.

Conditional Modulation of Biological Processes by Low-Temperature Degrons.

Conditional modulation of biological processes plays key roles in basic and applied research and in translation. It can be achieved on various levels via a multitude of approaches. One of the directions is manipulating target protein levels and activity by transcriptional, posttranscriptional, translational, and posttranslational control. Because in most of these techniques, the synthesis of the target proteins is adjusted to the needs, they all rely on the specific half-life of the target protein and its turn-over. Therefore, their time-of-action, in direct correlation to the desired reprogramming of molecular phenotypes caused by altering the target levels, is fixed and determined by the naturally inherent properties. We have introduced the low-temperature degron (lt-degron) to various intact multicellular organisms which allows to control target protein levels and therefore function and activity directly on the level of active protein. The lt-degron uses a combination of Ubiquitin-fusion technique linking target protein degradation to the N-end rule pathway of targeted proteolysis coupled with the use of cell- and tissue-specific promoters.

Direct Generation of Conditional Alleles Using CRISPR/Cas9 in Mouse Zygotes.

Conditional alleles in genetically modified mice allow for the deletion of a gene of interest in a target tissue when combined with a tissue-specific Cre recombinase. A conditional allele is achieved by introducing LoxP sites around a critical exon, a gene, or a cluster of genes. Previously, conditional alleles were introduced in the mouse germline by classic gene targeting in embryonic stem cells, a challenging and time-consuming procedure. Now, conditional alleles can be generated directly in fertilized mouse eggs (zygotes) using the CRISPR/Cas9 technology. This one-step generation of mice is easier in design and faster. Here, we describe our achieved success rate, the considerations in design of a conditional allele, a detailed protocol to prepare the zygote injection mix, and the screening procedure to identify the new conditional knockout mouse strain.

Conditional targeting of Ispd using paired Cas9 nickase and a single DNA template in mice.

CRISPR/Cas9 technology is a highly promising genome editing tool in the mouse, potentially overcoming the costs and time required for more traditional gene targeting methods in embryonic stem (ES) cells. Recently, compared to the wildtype nuclease, paired Cas9 nickase (Cas9n) combined with single guide RNA (sgRNA) molecules has been found to enhance the specificity of genome editing while reducing off-target effects. Paired Cas9n has been shown to be as efficient as Cas9 for generating insertion and deletion (indel) mutations by non-homologous end joining and targeted deletion in the genome. However, an efficient and reliable approach to the insertion of loxP sites flanking critical exon(s) to create a conditional allele of a target gene remains an elusive goal. In this study, we microinjected Cas9n RNA with sgRNAs together with a single DNA template encoding two loxP sites flanking (floxing) exon 2 of the isoprenoid synthase containing domain (Ispd) into the pronucleus and cytoplasm of C57BL/6NCr one-cell stage zygotes. After surgical transfer, one F0 mouse expressing a conditional allele was produced (at a frequency of ∼8% of live pups born). The floxed allele was transmitted through the germline to F1 progeny, and could be successfully recombined using Cre recombinase. This study indicates that conditional targeting can be accomplished effectively using paired Cas9n and a single DNA template.