Heritable Multiplex Genetic Engineering in Rats Using CRISPR/Cas9

<div><p>The CRISPR/Cas9 system has been proven to be an efficient gene-editing tool for genome modification of cells and organisms. Multiplex genetic engineering in rat holds a bright future for the study of complex disease. Here, we show that this system enables the simultaneous disruption of four genes (<i>ApoE</i>, <i>B2m</i>, <i>Prf1</i>, and <i>Prkdc</i>) in rats in one-step, by co-injection of Cas9 mRNA and sgRNAs into fertilized eggs. We further observed the gene modifications are germline transmittable, and confirmed the off-target mutagenesis and mosaicism are rarely detected by comprehensive analysis. Thus, the CRISPR/Cas9 system makes it possible to efficiently and reliably generate gene knock-out rats.</p></div

Echocardiography and H&E staining.

<p>TG mice and their NTG littermates were analyzed using M-mode echocardiographic analyses at 1, 3, 5, and 7 months of age. Fractional shortening (FS%) was decreased by the expression of HB-EGF compared with that of NTG mice (A). The left ventricular end-systolic diameter (LVIDs) was increased by the transgenic expression of HB-EGF compared with that of NTG mice (B), while left ventricular anterior wall thickness at end-systole (LVAWs) was not different (C). The whole heart (original magnification ×4) from TG and NTG littermates were sampled and stained using H&E and observed with a microscope. Cardiomyocytes (original magnification ×400) from the same areas of the left ventricle from TG and NTG littermates were compared to detect changes of myocyte cross-section size (D). About 50 cells per sample slice were randomly selected to calculate the average area of a single cell size (E, <i>n</i> = 4). *<i>P</i><0.05 <i>vs.</i> NTG mice; <i>P</i><0.001 <i>vs.</i> NTG mice.</p

Summary of the mutation of the founder rats.

<p>Single sgRNA/Gene: A mixture of 4 sgRNAs, each targeting a single site in each of the 4 genes. Dual sgRNAs/Gene: Another distinct sgRNA of each gene, together with the tested 4 sgRNAs targeting double site in each of the 4 genes.</p

Generation of TG mice.

<p>The HB-EGF TG construct was generated by the insertion of a target gene under the control of the α-MHC cardiac-specific promoter (A), and the TG mice were created using a microinjection method. Mouse lines with overexpression of HB-EGF (proHB-EGF and mature HB-EGF) were selected using a western immunoblot procedure and normalized with GAPDH (B).</p

Determination of cardiac fibrosis.

<p>Mice were sacrificed at 1, 3, 5 and 7 months of age, and heart tissues were examined for pathological changes and collagen expression. Heart fibrosis, as determined by Masson staining (A, original magnification ×200). The fibrosis area of the section was quantified using image-Pro Plus 6.0 software, the ratio of collagen area and total area (×%) was counted and compared (B). *<i>P</i><0.05 <i>vs.</i> NTG mice. The synthesis of Col1α1 and Col3α1 was detected using quantitative real-time PCR (C) and RT-PCR (D). <i>P</i><0.001 <i>vs.</i> Col1α1 of NTG mice; *<i>P</i><0.05 <i>vs.</i> Col3α1 of NTG mice; **<i>P</i><0.001 <i>vs.</i> Col3α1 of NTG mice. The Col1α1/Col3α1 ratio was compared between NTG and TG mice (E). *<i>P</i><0.05 <i>vs.</i> NTG mice; **<i>P</i><0.001 <i>vs.</i> NTG mice.</p

Heparin-Binding EGF-Like Growth Factor Induces Heart Interstitial Fibrosis via an Akt/mTor/p70s6k Pathway

<div><p>Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is essential for maintaining normal function of the adult heart and is known to play an important role in myocardial remodeling. In the present study, we observed that heart-specific HB-EGF transgenic (TG) mice had systolic dysfunction with decreased fractional shortening (FS%), increased end-systolic diameter (LVIDs) at 5 months of age, increased heart fibrosis, and increased mRNA expression of Col1α1 and Col3α1 at 1, 3, 5 and 7 months of age compared to nontransgenic (NTG) littermates. However, the left ventricular anterior wall thickness at end-systole (LVAWs) of the TG mice was not different than the NTG mice. Phosphorylation levels of Akt, mTor and p70s6k were increased due to HB-EGF expression in TG mice compared with the NTG mice at 3 and 7 months of age. Additionally, activated Akt, mTor and p70s6k were co-localized with vimentin to cardiac fibroblasts isolated from TG mice. Furthermore, HB-EGF significantly increased phosphorylation levels of Akt, mTor and p70s6k and increased expression of type I collagen in cultured primary cardiac fibroblasts. Rapamycin (Rapa) and CRM197, inhibitors of mTor and HB-EGF respectively, could inhibit the expression of type I collagen in the cultured primary cardiac fibroblasts and Rapa suppressed interstitial fibrosis of the heart tissues <em>in vivo</em>. In addition, a BrdU assay showed that HB-EGF increased proliferation of cardiac fibroblasts by 30% compared with cells without HB-EGF treatment. HB-EGF-induced proliferation was completely diminished in the presence of Rapa. These results suggest that HB-EGF induced heart fibrosis and proliferation of cardiac fibroblasts occurs through activation of the Akt/mTor/p70s6k pathway.</p> </div

CypA suppressed influenza virus replication at the post-transcription level.

<p>Relative quantitation of M1 mRNA (A), vRNA (B), and cRNA (C) in cells infected with A/WSN/33 at different time points post-infection. As described in the experimental procedures, different primers were used for the cDNA synthesis of mRNA, vRNA, and cRNA. Comparison of viral RNA levels from influenza A M1 genes in the presence or absence of CypA. The specificities of the amplified products were all confirmed by melting curve analysis. In all of the PCR assays, GAPDH was used as an internal control. Data are means ± SD of three separate experiments.</p

Determination of the phosphorylation levels of Akt, mTor and p70s6k in heart tissues.

<p>Mice were sacrificed at 3 and 7 months of age, and heart-tissue samples were homogenized in RIPA buffer. Phosphorylation levels of Akt, mTor and p70s6k were detected using western immunoblot (A). The relative intensities were detected using Image J and compared with those of NTG mice (B). *<i>P</i><0.05 <i>vs.</i> NTG mice at 3month; **<i>P</i><0.001 <i>vs.</i> NTG mice at 3month; †<i>P</i><0.05 <i>vs.</i> NTG mice at 7month; ‡<i>P</i><0.001 <i>vs.</i> NTG mice at 7month.</p

Localization of activated Akt, mTor and p70s6k in mouse heart tissues using double immunofluorescence confocal staining at 7 months of age.

<p>Cell localization of p-Akt (A), p-mTor (B) and p-p70s6k (C) were observed using confocal laser- scanning microscopy (Leica TCS SP2, Germany). Scale bar = 20 µm.</p

Generation of multiplex genetic modified rats using CRISPR/Cas9 system.

<p>(a) Detection of Cas9:sgRNA-mediated site-specific cleavage of the endogenous <i>ApoE</i>, <i>B2m</i>, <i>Prf1</i>, and <i>Prkdc</i> by T7EN1 cleavage assay. PCR amplicon of the targeted fragment at the <i>ApoE</i>, <i>B2m</i>, <i>Prf1</i>, and <i>Prkdc</i> in 15 founder rats (#1∼15) were subjected to T7EN1 cleavage assay. Founder #13, which is quadruple gene mutant, was marked with asterisks. (b) DNA sequences of four loci in founder #13. PCR amplicon with cleaved bands in T7EN1 cleavage assay were cloned and sequenced. The PAM sequence was underlined and highlighted in green; the targeting site are red; the mutations are blue, lower case; insertions (+) or deletions (−) are shown to the right of each allele.</p