The study of model organisms has revolutionized our understanding of the mechanisms underlying normal development, adult homeostasis, and human disease. Much of what we know about gene function in model organisms (and its application to humans) has come from gene knockouts: the ability to show analogous phenotypes upon gene inactivation in animal models. The zebrafish (Danio rerio) has become a popular model organism for many reasons, including the fact that it is amenable to various forms of genetic manipulation. The RNA-guided CRISPR/Cas9-mediated targeted mutagenesis approaches have provided powerful tools to manipulate the genome toward developing new disease models and understanding the pathophysiology of human diseases. CRISPR-based approaches are being used for the generation of both knockout and knock-in alleles, and also for applications including transcriptional modulation, epigenome editing, live imaging of the genome, and lineage tracing. Currently, substantial effort is being made to improve the specificity of Cas9, and to expand the target coverage of the Cas9 enzymes. Novel types of naturally occurring CRISPR systems [Cas12a (Cpf1); engineered variants of Cas9, such as xCas9 and SpCas9-NG], are being studied and applied to genome editing. Since the majority of pathogenic mutations are single point mutations, development of base editors to convert C:G to T:A or A:T to G:C has further strengthened the CRISPR toolbox. In this review, we provide an overview of the increasing number of novel CRISPR-based tools and approaches, including lineage tracing and base editing.

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Aminoacyl-tRNA synthetases (ARSs) are essential enzymes for faithful assignment of amino acids to their cognate tRNA. Variants in ARS genes are frequently associated with clinically heterogeneous phenotypes in humans and follow both autosomal dominant or recessive inheritance patterns in many instances. Variants in tryptophanyl-tRNA

Deafness, the most frequent sensory deficit in humans, is extremely heterogeneous with hundreds of genes involved. Clinical and genetic analyses of an extended consanguineous family with pre-lingual, moderate-to-profound autosomal recessive sensorineural hearing loss, allowed us to identify CLRN2, encoding a tetraspan protein, as a new deafness gene. Homozygosity mapping followed by exome sequencing identified a 14.96 Mb locus on chromosome 4p15.32p15.1 containing a likely pathogenic missense variant in CLRN2 (c.494C > A, NM_001079827.2) segregating with the disease. Using in vitro RNA splicing analysis, we show that the CLRN2 c.494C > A variant leads to two events: (1) the substitution of a highly conserved threonine (uncharged amino acid) to lysine (charged amino acid) at position 165, p.(Thr165Lys), and (2) aberrant splicing, with the retention of intron 2 resulting in a stop codon after 26 additional amino acids, p.(Gly146Lysfs*26). Expression studies and phenotyping of newly produced zebrafish and mouse models deficient for clarin 2 further confirm that clarin 2, expressed in the inner ear hair cells, is essential for normal organization and maintenance of the auditory hair bundles, and for hearing function. Together, our findings identify CLRN2 as a new deafness gene, which will impact future diagnosis and treatment for deaf patients.

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Powerful and simple, RNA-guided CRISPR/Cas9 technology is a versatile genome editing tool that has revolutionized targeted mutagenesis. cRiSpR-based genome editing has enabled large-scale functional genetic studies through the generation of gene knockouts in a variety of model organisms including zebrafish, and can be used to target multiple genes simultaneously. One of the challenges associated with the large scale application of this technique to zebrafish is the lack of a cost-effective method by which to identify mutants. to address this, we optimized the high-throughput, high-resolution fluorescent PCR-based fragment analysis method to develop MultiFRAGing -a robust and cost-effective method to genotype multiple targets in a single reaction. our approach can identify indels in up to four targets from a single reaction, which represents a four-fold increase in genotyping throughput. this method can be used by any laboratory with access to capillary electrophoresis-based sequencing equipment.Following completion of the human genome sequencing project, identification of candidate disease genes has been the focus of much genetic research. With the development of less expensive sequencing technologies, such genes are being discovered at a rapid rate, but functional validation remains slow. Most of the knowledge of gene function has been generated using gene knockout technology in model organisms 1 .The zebrafish (Danio rerio) has become a popular model organism for many reasons including high fecundity, optically transparent embryos and larvae, external development and the ease with which various types of genetic manipulation can be performed 2 . A large number of zebrafish mutants have been generated using a variety of random mutagenesis approaches involving either the chemical mutagen N-ethyl-N-nitrosourea (ENU) or insertional mutagens (Retroviruses, Transposons) 2 . Recent progress in the transformative, targeted, and simple RNA-guided CRISPR/Cas9-based genome editing method has expedited genetic manipulation in many systems, including zebrafish 3-5 . In addition to CRISPR/Cas9, Transcription Activator-like Effector Nuclease(TALENS), and Zinc Finger Nucleases (ZFNs) are other targeted mutagenesis methods being used to generate knockouts for the purposes of developing disease models and understanding disease pathology in zebrafish 2,6 . Recently, the structure-guided endonuclease (SGN) -a DNA-guided genome editing tool that uses flap endonuclease 1 (FEN-1) fused to the Fok1 endonuclease -was also used to generate large deletions in the zebrafish genome 7,8 . While CRISPR/Cas9 is a RNA-guided endonuclease and TALENs and ZFNSs are engineered proteins, the SGN nuclease functions in a similar manner: it induces a double stranded break (DSB) at a DNA target site which is then repaired by an error-prone, non-homologous end-joining method that often leaves insertions and/or deletions during the repair.Because of its simplicity, low-cost and ability to target multiple sites simultaneously, CRISPR/Cas9 is becoming most ...

Front Cover: The cover image is based on the Research Article Biallelic variants in WARS1 cause a highly variable neurodevelopmental syndrome and implicate a critical exon for normal auditory function by Sheng‐Jia Lin et al., https://doi.org/10.1002/humu.24435.

The powerful and simple RNA-guided CRISPR/Cas9 technology is a versatile genome editing tool that has revolutionized targeted mutagenesis. CRISPR-based genome editing has enabled large-scale functional genetic studies through the generation of gene knockouts in a variety of model organisms including zebrafish. CRISPR/Cas9 can also be used to target multiple genes simultaneously. One of the challenges associated with applying this technique to zebrafish in a high-throughput manner is the absence of a cost-effective method by which to identify mutants. To address this, we optimized the high-throughput, high-resolution fluorescent PCR-based fragment analysis method to develop MultiFRAGing, a robust and cost-effective method for genotyping of multiple targets in a single reaction. Our approach can identify indels in 4 targets from a single reaction, which represents a four-fold increase in genotyping throughput. This method can be used by any laboratory with access to capillary electrophoresis based sequencing equipment.