CRISPR/Cas9 technology-mediated genome editing has significantly improved the targeted inactivation of genes in vitro and in vivo in many organisms. In this study, we have reported a novel CRISPR-based vector system for conditional tissue-specific gene ablation in zebrafish. Specifically, the cardiac-specific cardiac myosins light chain 2 (cmlc2) promoter drives Cas9 expression to silence the neuropilin-1(nrp1) gene in cardiomyocytes in a heat-shock inducible manner. This vector system establishes a unique tool to regulate the gene knockout in both the developmental and adult stages and hence, widens the possibility of loss-of-function studies in zebrafish at different stages of development and adulthood. Using this approach, we investigated the role of neuropilin isoforms nrp1a and nrp1b in response to cardiac injury and regeneration in adult zebrafish hearts. We observed that both the isoforms (nrp1a and nrp1b) are upregulated after the cryoinjury. Interestingly, the nrp1b-knockout significantly altered heart regeneration and impaired cardiac function in the adult zebrafish, demonstrated by reduced heart rate (ECG), ejection fractions, and fractional shortening. In addition, we show that the knockdown of nrp1b but not nrp1a induces activation of the cardiac remodeling genes in response to cryoinjury. To our knowledge, this is the first study where we have reported a heat shock-mediated conditional knockdown of nrp1a and nrp1b isoforms using CRISPR/Cas9 technology in the cardiomyocyte in zebrafish, and furthermore have identified a crucial role for nrp1b isoform in zebrafish cardiac remodeling and eventually heart function in response to injury.

The authors have declared no competing interest.

Analyzing a person’s gene expression requires mapping their RNA landscape to a standard reference to gain insight into the degree to which genes are “turned on” and perform functions in the body. But researchers can run into issues when the reference does not provide enough information to allow for accurate mapping, an issue known as reference bias.

In a new paper published in the journal Nature Methods, researchers at UC Santa Cruz introduce the first-ever method for analyzing RNA sequencing data genome-wide using a “pantranscriptome,” which combines a transcriptome and a pangenome—a reference that contains genetic material from a cohort of diverse individuals, rather than just a single linear strand.

A group of scientists led by UCSC Associate Professor of Biomolecular Engineering Benedict Paten have released a toolkit that allows researchers to map an individual’s RNA data to a much richer reference, addressing reference bias and leading to much more accurate mapping.

Many genetic diseases are caused by diverse mutations spread across an entire gene, and designing genome editing approaches for each patient’s mutation would be impractical and costly.

Investigators at Massachusetts General Hospital (MGH) have recently developed an optimized method that improves the accuracy of inserting large DNA segments into a genome.

This approach could be used to insert a whole normal or “wild-type” replacement gene, which could act as a blanket therapy for a disease irrespective of a patient’s particular mutation.