Generation of a New Tau Knockout (tau Δex1 ) Line Using CRISPR/Cas9 Genome Editing in Mice

INTRODUCTION

The tau protein belongs to a family of microtubule-associated proteins (MAPs) and is found at high levels in neurons. In humans, tau is encoded by the microtubule-associate protein tau (MAPT) gene on chromosome 17, while in mice, the corresponding Mapt gene is located on chromosome 11. The MAPT mRNA is subject to alternative splicing of exons 2, 3, and 10, giving rise to 6 different isoforms found at equal levels in the adult human brain, and containing either 0, 1, or 2 amino-terminal inserts (encoded by exons 2 and 3) and 3 or 4 microtubule-binding repeats (3/4R) in its carboxy-terminal half [1]. During embryonic development in both humans and mice, 3R tau isoforms are predominant. The adult mouse brain differs from the human brain, in that only 4R tau isoforms are present shortly after birth [2, 3]. Functionally, tau contributes to dynamics of the microtubule cytoskeleton and regulates cargo transport along neuronal processes [4]. We have shown in addition that tau has important functions in regulating post-synaptic signaling processes [5–7].

Tau rose to fame when it was discovered as the major constituent of neurofibrillary tangles in Alzheimer’s disease (AD), where it undergoes aberrant hyperphosphorylation [8]. Furthermore, tau becomes hyperphosphorylated and forms intracellular deposits in a range of frontotemporal dementia (FTD) subtypes [9]. Familial forms of FTD have been linked to pathogenic mutations in MAPT [10]. Accordingly, transgenic mice expressing mutant human tau have contributed to the understanding of disease mechanism in AD and FTD [11, 12]. Conversely, tau-deficient mice (tau^–/–) have been generated to study its role in disease and physiology [13]. Interestingly, tau^–/– mice initially did not present with overt deficits [14, 15], possibly due to compensation by other MAPs [16]. More detailed testing revealed specific deficits in some tau^–/– lines as they age [17–20], while others did not report functional impairments even at advanced ages [6, 21–23]. A critical role in the early pathogenesis of AD was established by showing that amyloid-β-induced deficits were prevented on a tau^–/– background [7, 24], although one report showed enhanced pathology under similar experimental conditions [25]. It has been suggested that the conflicting findings from tau^–/– mice may be due to variable genetic background and/or differences in holding and nutrition of the mice [23]. Furthermore, all tau^–/– lines characterized so far have been generated by ES-cell gene targeting, which relies on the insertion of large transgenes in the coding part of the Mapt gene to disrupt its expression [13]. The insertion of large selectable marker cassettes (typically after gene trap experiments) can create phenocopy and/or unwanted confounding effects [26–28] and create inconsistent results.

Here, we report the generation of a new tau^–/– resource by targeting the transcriptional start codon in exon 1 of Mapt via the introduction of a short deletion (tau^Δex1). The tau^Δex1 line was generated on a pure C57Bl/6J background and reproduced resistance to excitotoxicity in the absence of memory deficits in young mice, reported for earlier tau^–/– lines [7, 21].

MATERIALS AND METHODS

RESULTS AND DISCUSSION

We have previously used tau^–/– mice that were generated by knocking in a GFP-encoding cDNA into exon 1 of Mapt using homologous recombination in embryonic stem cells and blastocyst injection [31]. Homozygous mice lacked tau expression, and instead expressed a fusion protein comprising the first 31 amino acids of tau followed by GFP. GFP expression limited functional studies using multiple fluorescence channels. Furthermore, previous tau^–/– lines, including the GFP knockin line, were generated on mixed genetic backgrounds and required introduction of selection cassettes into the genome [13].

In the present study, we aimed at generating tau^–/– mice with a small genome alteration directly on a C57Bl/6J background to study the function of tau in subsequent projects. In order to introduce a small deletion at the intron -1/exon 1 junction of Mapt by CRISPR/Cas9 genome editing we designed two RNA guides, 115 bp up- and 18 bp down-stream of the transcriptional start codon (Fig. 1A). Guides and Cas9 protein were injected into the cytoplasm of fertilized C57Bl/6 oocytes. All offspring were genotyped for the presence of a deletion in Mapt (Fig. 1B). Out of two offspring, two carried homozygous deletion. A male founder with an approximately 150 bp deletion, based on the size difference of genotyping bands, was chosen to establish a tau^Δex1 line by crossing with C57Bl/6J mice. A heterozygous tau^Δex1 offspring of this first generation (F1) was in turn crossed with C57Bl/6J mice, before intercrossing heterozygous tau^Δex1 F2 offspring to obtain homozygous tau^Δex1 mice. This crossing scheme ensured that possible differences in the initial deletion of Mapt on the two alleles were not carried through to subsequent generations. Breeding performance of tau^Δex1 mice was not compromised and genetic modifications were inherited at Mendelian ratios across generations. Tau^Δex1 mice showed no overt phenotype, and were indistinguishable from their wild-type littermates in weight and size.

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Fig.1

Generation of tau^Δex1 mice. A) Schematic of MAPT targeting strategy. The Cas9/sgRNA-targeting sequences are underlined and the protospacer-adjacent motif (PAM) sequences are in red. Arrows indicate the position of primers used for sequencing. B) Genotyping of homozygous (–/–) and heterozygous (+/–) tau^Δex1 mice and wild-type littermates (+/+); 666 bp band indicates MAPT wild-type allele and 527 bp bands the deletion allele. C) Sequencing result of the mutant MAPT allele in homozygous tau^Δex1 mice confirms deletion of a 139 bp sequences including the ATG start codon (underlined), between the sgRNA sequences (boxed). D) Western blotting of brain extracts from homozygous (–/–) and heterozygous (+/–) tau^Δex1 mice and wild-type littermates (+/+), showing reduction and loss of tau expression in heterozygous (+/–) and homozygous (–/–) tau^Δex1 mice, respectively. Levels of MAP1A, MAP1B, and MAP2, however, were comparable in homozygous (–/–) and heterozygous (+/–) tau^Δex1 mice and wild-type littermates (+/+). Quantification is shown from independent experiments (n = 6; *p < 0.05; ****p < 0.0001 [One-way ANOVA]).

To establish the exact size of the introduced deletion, we amplified a 666 bp fragment from homozygous tau^Δex1 mice using primers located up- and down-stream of the predicted site in Mapt. The resulting PCR fragment was sequenced, confirming a 139 bp deletion, comprising 106 bp in the intron immediately up-stream of exon 1, plus the first 33 bp of exon 1 itself (Fig. 1C). This deletion included the transcription start codon in exon 1 of Mapt. To confirm that the deletion resulted in reduction or absence of tau protein in hetero- and homozygous tau^Δex1 mice, respectively, we performed immunoblotting of brain extracts using tau-specific antibodies. This confirmed significant reduction of tau expression in heterozygous tau^Δex1 mice and absence of tau protein in homozygous tau^Δex1 mice (Fig. 1D). Compensatory upregulation of Map1A has been reported in one previous tau^–/– strain [14]. Therefore, we also probed immunoblots for other MAP family members, including Map1A, Map1B, and Map2. We did not find changes in any of those MAP family members at 6 weeks of age (Fig. 1D). While this suggests no general compensation for the loss of tau by upregulating other MAP family members, a temporal and spatial profiling of MAP expression levels and subcellular distribution would be required to determine earlier or later changes in MAP in tau^Δex1 neurons.

Others and we have previously reported that different tau^–/– lines display an increased resistance to induced excitotoxic seizures [7, 21]. To confirm that this robust phenotype is also presented by our new strain, we subjected both hetero- and homozygous tau^Δex1 mice and their non-mutant littermates to induced excitotoxic seizures at 6 weeks of age. Administration of PTZ (50 mg/kg i.p.) resulted in severe seizures in wild-type littermates (Fig. 2A). In contrast, the mean seizure severity was significantly reduced in heterozygous and, to a greater extend, homozygous tau^Δex1 mice. Furthermore, the latency to develop more severe seizures was increased in homozygous tau^Δex1 mice compared to heterozygous tau^Δex1 mice and wild-type littermates for seizures of minimal severity (score 1–2) (Fig. 2B). Notably, 31% of homozygous tau^Δex1 mice developed bouncing seizures (score 5) without showing prior forelimb or generalized clonus (scores 3–4) and only 6% presenting prior tail expansion (score 2) (Fig. 2C). Forty-five percent of heterozygous tau^Δex1 mice developed bouncing seizures (score 5), but showed prior progression with tail extension, forelimb and generalized clonus. In comparison, bouncing seizures were observed in 80% of the wild-type littermates, with 27% and 7% reaching more severe body extension and (non-recover) status epilepticus, respectively. Taken together, tau^Δex1 presented with a similar reduction in susceptibility to induced excitotoxic seizures, as did previous tau^–/– strains [7, 21].

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Fig.2

Decreased susceptibility to induced excitotoxic seizures in tau^Δex1 mice. A) Mean seizure severity was significantly reduced in 6-weeks-old homozygous (–/–) and heterozygous (+/–) tau^Δex1 mice compared to wild-type littermates (+/+) (n = 16 (–/–), n = 11 (+/–), n = 15 (+/+); *p < 0.05; ****p < 0.0001 [One-way ANOVA]). B) Homozygous tau^Δex1 mice (–/–) showed delayed latency to develop mild seizures, while seizure progression to other stages was comparable to heterozygous (+/–) tau^Δex1 mice and wild-type littermates (+/+). C) Percentage of mice reaching different seizure scores. Less heterozygous (+/–) tau^Δex1 mice reached higher seizure severity scores than wild-type littermates (+/+). Only few homozygous tau^Δex1 mice (–/–) progressed to even mild seizure severity, and some to seizure score 5.

Others and we have previously reported that depletion of tau has no impact on memory formation in young [6, 21] and aged [23] tau^–/– mice, while again others reported deficits in aged cohorts [19]. To determine learning in tau^Δex1 mice, we subjected 4-month-old animals to a standard Morris water maze paradigm and assessed the decrease in latency to find the submerged platform over subsequent testing days as an indicator of memory formation. To compare the performance of tau^Δex1 to previous tau^–/– mice, we included GFP knockin tau^–/– (tau^eGFP) mice [6, 31] in these experiments. As expected, the escape latency decreased similarly over time for all genotypes, indicative of normal learning (Fig. 3A). Hence, learning was normal in young tau^Δex1 mice. To determine whether there are differences in memory formation in the absence of tau, we analyzed the change in exploration strategy of individual mice over time. Following Garthe and Kempermann [35], swim traces from individual sessions were scored according to their strategy to find the submerged platform, ranging from thigmotaxis (score 1) to a direct swim path (score 7) (Fig. 3B). Irrespective of the genotype, the majority of mice showed random swim patterns upon test commencement on Day 1 (Fig. 3C). Interestingly, a larger number of homozygous tau^eGFP mice showed thigmotaxis on the first two testing days compared to tau^Δex1 and tau^+/+ mice. As testing progressed, random swimming was replaced by more successful strategies. Although by Day 5, similar proportions of homozygous tau^Δex1 and tau^eGFP, and tau^+/+ mice had developed goal-targeted swim strategies (scores 5 to 7), tau^eGFP maintained non-goal-targeted swim strategies (scores 3 and 4) for longer than homozygous tau^Δex1 and tau^+/+ mice. Taken together, absence of tau did not affect memory formation in adult mice. Future studies with aged mice are required to determine memory formation as tau^Δex1 mice age.

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Fig.3

Normal memory formation in tau^Δex1 mice. A) Four-month-old homozygous tau^Δex1 mice (–/–; n = 7), homozygous eGFP tau knockin (tau^egfp; n = 10) and wild-type (tau+/+; n = 11) mice were tested in the Morris water maze paradigm. Comparably improved latency to locate the submerged platform in all mice suggests normal memory formation. B) A schematic representation of the 7 search strategies observed in swim paths in the Morris water maze including color coding and score for each strategy. C) Prevalence of each strategy were similar in homozygous (–/–) and heterozygous (+/–) tau^Δex1 mice compared to wild-type littermates (+/+), indicating comparable proficiency in spatial task.

In summary, we have generated a new tau^–/– line, tau^Δex1, by CRISPR/Cas9-mediated deletion of the transcriptional start in exon 1 on a pure C57Bl/6J background. Tau^Δex1 mice had no overt phenotypes, including normal learning in a standard test paradigm, but showed reduced susceptibility to excitotoxicity, which is in line with previous reports of other tau^–/– lines [5–7, 21]. We anticipate that tau^Δex1 mice will contribute to unraveling new functions of tau in physiology and disease, and may help in understanding differences between previously reported tau^–/– strains. To facilitate this, we will make tau^Δex1 mice available without restrictions.