CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells

Clustered interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome engineering technologies are sparking a new revolution in biological research. This technology efficiently induces DNA double strand breaks at the targeted genomic sequence and results in indel mutations by the error-prone process of nonhomologous end joining DNA repair or homologous recombination with a DNA repair template. The efficiency of genome editing with CRISPR/Cas9 alone in human embryonic stem cells is still low. Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types with maximal targeting frequencies without engineered nucleases. However, whether CRISPR/Cas9-mediated double strand breaks and AAV based donor DNA mediated homologous recombination approaches could be combined to create a novel CRISPR/Cas9-AAV genetic tool for highly specific gene editing is not clear. Here we demonstrate that using CRISPR/Cas9-AAV, we could successfully knock-in a DsRed reporter gene at the basic motifleucine zipper transcription factor (NRL) locus in human embryonic stem cells. For the first time, this study provides the proof of principle that these two technologies can be used together. CRISPR/Cas9-AAV, a new genome editing tool, offers a platform for the manipulation of human genome

CRISPR/Cas9-loxP-Mediated Gene Editing as a Novel Site-Specific Genetic Manipulation Tool

Cre-loxP, as one of the site-specific genetic manipulation tools, offers a method to study the spatial and temporal regulation of gene expression/inactivation in order to decipher gene function. CRISPR/Cas9-mediated targeted genome engineering technologies are sparking a new revolution in biological research. Whether the traditional site-specific genetic manipulation tool and CRISPR/Cas9 could be combined to create a novel genetic tool for highly specific gene editing is not clear. Here, we successfully generated a CRISPR/Cas9-loxP system to perform gene editing in human cells, providing the proof of principle that these two technologies can be used together for the first time. We also showed that distinct non-homologous end-joining (NHEJ) patterns from CRISPR/Cas9-mediated gene editing of the targeting sequence locates at the level of plasmids (episomal) and chromosomes. Specially, the CRISPR/Cas9-mediated NHEJ pattern in the nuclear genome favors deletions (64%–68% at the human AAVS1 locus versus 4%–28% plasmid DNA). CRISPR/Cas9-loxP, a novel site-specific genetic manipulation tool, offers a platform for the dissection of gene function and molecular insights into DNA-repair pathways

Recapitulating X-Linked Juvenile Retinoschisis in Mouse Model by Knock-In Patient-Specific Novel Mutation

X-linked juvenile retinoschisis (XLRS) is a retinal disease caused by mutations in the gene encoding retinoschisin (RS1), which leads to a significant proportion of visual impairment and blindness. To develop personalized genome editing based gene therapy, knock-in animal disease models that have the exact mutation identified in the patients is extremely crucial, and that the way which genome editing in knock-in animals could be easily transferred to the patients. Here we recruited a family diagnosed with XLRS and identified the causative mutation (RS1, p.Y65X), then a knock-in mouse model harboring this disease-causative mutation was generated via TALEN (transcription activator-like effector nucleases). We found that the b-wave amplitude of the ERG of the RS1-KI mice was significantly decreased. Moreover, we observed that the structure of retina in RS1-KI mice has become disordered, including the disarray of inner nuclear layer and outer nuclear layer, chaos of outer plexiform layer, decreased inner segments of photoreceptor and the loss of outer segments. The novel knock-in mice (RS1-KI) harboring patient-specific mutation will be valuable for development of treatment via genome editing mediated gene correction

T148 is a well-conserved amino acid of Cx46 and <i>GJA3</i>/p. T148I is a novel mutation with co-segregation in this family.

<p>(A) <i>GJA3</i>/p. T148I is located at the cytoplasmic loop (indicated by the blue square) domain of the Cx46 protein. The membrane topological structure of Cx46 was generated by TOPO2 software. This mutation (indicated by the red square) is located in the cytoplasmic loop domain; the black square indicates reported mutations associated with CCs (E: extracellular; M: membrane; I: intracellular). (B) Sanger sequencing results showed that this missense mutation was detected in all affected individuals and was not shown in unaffected family members or in the 100 unrelated control individuals. (C) Multiple protein sequence alignments. Multiple sequence alignments of <i>GJA3</i> from different species and <i>GJA</i> family members (including <i>GJA1</i>, <i>GJA5</i>, and <i>GJA8</i>) from a human revealed that codon 148, where the mutation (p. T148I) occurred, was located within a highly conserved region. The “mut.” sequence indicates the sequence with the mutation detected in this family.</p

Fluorescence microscopy and Western blot analysis.

<p>(A) pEGFP-N1, Cx46WT, Cx46T148I and Cx46G143R EGFP-tagged proteins were located in the cytoplasm and plasma membrane of normal-density HLECs. However, Cx46T148I as well as the reported Cx46-G143R showed aggregate signals from cytoplasmic inclusions in fluorescent images rather than the typical punctate staining in the WT proteins. (B) Cx46T148I and Cx46-G143R significantly increased the intracellular aggregation. (C) and (D) Western blot analysis of the cell lysates indicated a higher expression level of mutant Cx46/Flag than the WT or control lanes. Scale bar:20 μm.</p

Hydrophilicity analysis of the WT and mutant proteins as well as homologous modeling predicted by the Swiss-Model program.

<p>(A) The hydropathic character of the changes in the mutant protein indicated a higher hydrophobicity than that of the WT. (B) The conformation of mutant Cx46 underwent a significant change with an α-helix deletion in the C-terminal when compared with that of the WT model. The α-helix deletion was marked in red circle and the locations of amino acid 148 were marked in red.</p

Photographs of affected individuals in this family.

<p>(A) the proband (III:1), (B) her older brother (III:3) and (C) the proband’s son (IV:5) showed bilateral pulverulent nuclear cataracts.</p

Pedigree of this ADCC family.

<p>Cataract pedigree. Squares and circles symbolize males and females, respectively. Black and white lines denote affected status and unaffected status, respectively. A solid black arrow indicates the proband (III:1), and the diagonal lines indicate deceased family members.</p

Identification of a novel <i>GJA3</i> mutation in a large Chinese family with congenital cataract using targeted exome sequencing

<div><p>Autosomal dominant congenital cataract (ADCC) is a clinically and genetically heterogeneous ocular disease in children that results in serious visual impairments or even blindness. Targeted exome sequencing (TES) is an efficient method used for genetic diagnoses of inherited diseases. In the present study, we used a custom-made TES panel to identify the genetic defect of a four-generation Chinese family with bilateral pulverulent nuclear cataracts. A novel heterozygous missense mutation c.443C>T (p. T148I) in <i>GJA3</i> was identified. The results of the bioinformatic analysis showed that the mutation was deleterious to the structure and hemichannel function of Cx46 encoded by <i>GJA3</i>. Plasmids expressing wild-type and mutant human Cx46 were constructed and ectopically expressed in human lens epithelial cells (HLECs) or human embryonic kidney (HEK-293) cells. Fluorescent images indicated aggregated signals of mutant protein in the cytoplasm, and a higher protein level was also detected in T148I stable cell lines. In summary, we identified a novel mutation in <i>GJA3</i> for ADCC, which provided molecular insights into the pathogenic mechanism of ADCC.</p></div