Establishment of human corneal epithelial organoids for ex vivo modelling dry eye disease.

Dry eye disease (DED) is a growing public health concern affecting millions of people worldwide and causing ocular discomfort and visual disturbance. Developing its therapeutic drugs based on animal models suffer from interspecies differences and poor prediction of human trials. Here, we established long-term 3D human corneal epithelial organoids, which recapitulated the cell lineages and gene expression signature of the human corneal epithelium. Organoids can be regulated to differentiate ex vivo, but the addition of FGF10 inhibits this process. In the hyperosmolar-induced DED organoid model, the release of inflammatory factors increased, resulting in damage to the stemness of stem cells and a decrease in functional mucin 1 protein. Furthermore, we found that the organoids could mimic clinical drug treatment responses, suggesting that corneal epithelial organoids are promising candidates for establishing a drug testing platform ex vivo. In summary, we established a functional, long-term 3D human epithelial organoid that may serve as an ex vivo model for studying the functional regulation and disease modelling.

Membrane Fusion-Mediated Loading of Therapeutic siRNA into Exosome for Tissue-Specific Application.

Tissue-specific delivery of oligonucleotide therapeutics beyond the liver remains a key challenge in nucleic acid drug development. To address this issue, exploiting exosomes as a novel carrier has emerged as a promising approach for efficient nucleic acid drug delivery. However, current exosome-based delivery systems still face multiple hurdles in their clinical applications. Herein, this work presents a strategy for constructing a hybrid exosome vehicle (HEV) through a DNA zipper-mediated membrane fusion approach for tissue-specific siRNA delivery. As a proof-of-concept, this work successfully fuses a liposome encapsulating anti-NFKBIZ siRNAs with corneal epithelium cell (CEC)-derived exosomes to form a HEV construct for the treatment of dry eye disease (DED). With homing characteristics inherited from exosomes, the siRNA-bearing HEV can target its parent cells and efficiently deliver the siRNA payloads to the cornea. Subsequently, the NFKBIZ gene silencing significantly reduces pro-inflammatory cytokine secretions from the ocular surface, reshapes its inflammatory microenvironment, and ultimately achieves an excellent therapeutic outcome in a DED mouse model. As a versatile platform, this hybrid exosome with targeting capability and designed therapeutic siRNAs may hold great potential in various disease treatments.

Safeguarding genome integrity during gene-editing therapy in a mouse model of age-related macular degeneration.

Ensuring genome safety during gene editing is crucial for clinical translation of the high-efficient CRISPR-Cas9 toolbox. Therefore, the undesired events including chromosomal translocations, vector integrations, and large deletions arising during therapeutic gene editing remain to be adequately addressed or tackled in vivo. Here, we apply CRISPR-Cas9TX in comparison to CRISPR-Cas9 to target Vegfa for the treatment of age-related macular degeneration (AMD) disease in a mouse model. AAV delivery of both CRISPR-Cas9 and CRISPR-Cas9TX can efficiently inhibit laser-induced neovascularization. Importantly, Cas9TX almost eliminates chromosomal translocations that occur at a frequency of approximately 1% in Cas9-edited mouse retinal cells. Strikingly, the widely observed AAV integration at the target Vegfa site is also greatly reduced from nearly 50% of edited events to the background level during Cas9TX editing. Our findings reveal that chromosomal structural variations routinely occur during in vivo genome editing and highlight Cas9TX as a superior form of Cas9 for in vivo gene disruption.

Comprehensive assessment of miniature CRISPR-Cas12f nucleases for gene disruption.

Because of their small size, the recently developed CRISPR-Cas12f nucleases can be effectively packaged into adeno-associated viruses for gene therapy. However, a systematic evaluation of the editing outcomes of CRISPR-Cas12f is lacking. In this study, we apply a high-throughput sequencing method to comprehensively assess the editing efficiency, specificity, and safety of four Cas12f proteins in parallel with that of Cas9 and two Cas12a proteins at multiple genomic sites. Cas12f nucleases achieve robust cleavage at most of the tested sites and mainly produce deletional fragments. In contrast, Cas9 and Cas12a show relatively higher editing efficiency at the vast majority of the tested sites. However, the off-target hotspots identified in the Cas9- and Cas12a-edited cells are negligibly detected in the Cas12f-edited cells. Moreover, compared to Cas9 and Cas12a nucleases, Cas12f nucleases reduce the levels of chromosomal translocations, large deletions, and integrated vectors by 2- to 3-fold. Therefore, our findings confirm the editing capacity of Cas12f and reveal the ability of this nuclease family to preserve genome integrity during genome editing.

PEM-seq comprehensively quantifies DNA repair outcomes during gene-editing and DSB repair.

The repair products of double-stranded DNA breaks (DSBs) are crucial for investigating the mechanism underlying DNA damage repair as well as evaluating the safety and efficiency of gene-editing; however, a comprehensively quantitative assay remains to be established. Here, we describe the step-by-step instructions of the primer extension-mediated sequencing (PEM-seq), followed by the framework of data processing and statistical analysis. PEM-seq presents a full spectrum of repair outcomes for both genome-editing-induced and endogenous DSBs in mouse and human cells. For complete details on the use and execution of this profile, please refer to Gan et al. (2021), Yin et al. (2019), Liu et al. (2021a), and Zhang et al. (2021).

Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing.

The mechanism underlying unwanted structural variations induced by CRISPR-Cas9 remains poorly understood, and no effective strategy is available to inhibit the generation of these byproducts. Here we find that the generation of a high level of translocations is dependent on repeated cleavage at the Cas9-targeting sites. Therefore, we employ a strategy in which Cas9 is fused with optimized TREX2 to generate Cas9TX, a Cas9 exo-endonuclease, which prevents perfect DNA repair and thereby avoids repeated cleavage. In comparison with CRISPR-Cas9, CRISPR-Cas9TX greatly suppressed translocation levels and enhanced the editing efficiency of single-site editing. The number of large deletions associated with Cas9TX was also reduced to very low level. The application of CRISPR-Cas9TX for multiplex gene editing in chimeric antigen receptor T cells nearly eliminated deleterious chromosomal translocations. We report the mechanism underlying translocations induced by Cas9, and propose a general strategy for reducing chromosomal abnormalities induced by CRISPR-RNA-guided endonucleases.

Global detection of DNA repair outcomes induced by CRISPR-Cas9.

CRISPR-Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive. We developed a new in silico analysis pipeline for the previously described primer-extension-mediated sequencing assay to comprehensively characterize CRISPR-Cas9-induced DSB repair outcomes in human or mouse cells. We identified tremendous deleterious DSB repair byproducts of CRISPR-Cas9 editing, including large deletions, vector integrations, and chromosomal translocations. We further elucidated the important roles of microhomology, chromosomal interaction, recurrent DSBs, and DSB repair pathways in the generation of these byproducts. Our findings provide an extra dimension for genome editing safety besides off-targets. And caution should be exercised to avoid not only off-target damages but also deleterious DSB repair byproducts during genome editing.

In-depth assessment of the PAM compatibility and editing activities of Cas9 variants.

A series of Cas9 variants have been developed to improve the editing fidelity or targeting range of CRISPR-Cas9. Here, we employ a high-throughput sequencing approach primer-extension-mediated sequencing to analyze the editing efficiency, specificity and protospacer adjacent motif (PAM) compatibility of a dozen of SpCas9 variants at multiple target sites in depth, and our findings validate the high fidelity or broad editing range of these SpCas9 variants. With regard to the PAM-flexible SpCas9 variants, we detect significantly increased levels of off-target activity and propose a trade-off between targeting range and editing specificity for them, especially for the near-PAM-less SpRY. Moreover, we use a deep learning model to verify the consistency and predictability of SpRY off-target sites. Furthermore, we combine high-fidelity SpCas9 variants with SpRY to generate three new SpCas9 variants with both high fidelity and broad editing range. Finally, we also find that the existing SpCas9 variants are not effective in suppressing genome instability elicited by CRISPR-Cas9 editing, raising an urgent issue to be addressed.

Pan-cancer analysis identifies telomerase-associated signatures and cancer subtypes.

BACKGROUND: Cancer cells become immortalized through telomere maintenance mechanisms, such as telomerase reverse transcriptase (TERT) activation. In addition to maintaining telomere length, TERT activates manifold cell survival signaling pathways. However, telomerase-associated gene signatures in cancer remain elusive. METHODS: We performed a systematic analysis of TERT high (TERThigh) and low (TERTlow) cancers using multidimensional data from The Cancer Genome Atlas (TCGA). Multidimensional data were analyzed by propensity score matching weight algorithm. Coexpression networks were constructed by weight gene coexpression network analysis (WGCNA). Random forest classifiers were generated to identify cancer subtypes. RESULTS: The TERThigh-specific mRNA expression signature is associated with cell cycle-related coexpression modules across cancer types. Experimental screening of hub genes in the cell cycle module suggested TPX2 and EXO1 as potential regulators of telomerase activity and cell survival. MiRNA analysis revealed that the TERThigh-specific miR-17-92 cluster can target biological processes enriched in TERTlow cancer and that its expression is negatively correlated with the tumor/normal telomere length ratio. Intriguingly, TERThigh cancers tend to have mutations in extracellular matrix organization genes and amplify MAPK signaling. By mining the clinical actionable gene database, we uncovered a number of TERThigh-specific somatic mutations, amplifications and high expression genes containing therapeutic targets. Finally, a random forest classifier integrating telomerase-associated multi-omics signatures identifies two cancer subtypes showed profound differences in telomerase activity and patient survival. CONCLUSIONS: In summary, our results depict a telomerase-associated molecular landscape in cancers and provide therapeutic opportunities for cancer treatment.