Multiplexed CRISPR/Cas9-mediated knockout of 19 Fanconi anemia pathway genes in zebrafish revealed their roles in growth, sexual development and fertility

Abstract

Fanconi Anemia (FA) is a genomic instability syndrome resulting in aplastic anemia, developmental abnormalities, and predisposition to hematological and other solid organ malignancies. Mutations in genes that encode proteins of the FA pathway fail to orchestrate the repair of DNA damage caused by DNA interstrand crosslinks. Zebrafish harbor homologs for nearly all known FA genes. We used multiplexed CRISPR/Cas9-mediated mutagenesis to generate loss-of-function mutants for 17 FA genes: fanca, fancb, fancc, fancd1/brca2, fancd2, fance, fancf, fancg, fanci, fancj/brip1, fancl, fancm, fancn/palb2, fanco/rad51c, fancp/slx4, fancq/ercc4, fanct/ube2t, and two genes encoding FA-associated proteins: faap100 and faap24. We selected two indel mutations predicted to cause premature truncations for all but two of the genes, and a total of 36 mutant lines were generated for 19 genes. Generating two independent mutant lines for each gene was important to validate their phenotypic consequences. RT-PCR from homozygous mutant fish confirmed the presence of transcripts with indels in all genes. Interestingly, 4 of the indel mutations led to aberrant splicing, which may produce a different protein than predicted from the genomic sequence. Analysis of RNA is thus critical in proper evaluation of the consequences of the mutations introduced in zebrafish genome. We used fluorescent reporter assay, and western blots to confirm loss-of-function for several mutants. Additionally, we developed a DEB treatment assay by evaluating morphological changes in embryos and confirmed that homozygous mutants from all the FA genes that could be tested (11/17), displayed hypersensitivity and thus were indeed null alleles. Our multiplexing strategy helped us to evaluate 11 multiple gene knockout combinations without additional breeding. Homozygous zebrafish for all 19 single and 11 multi-gene knockouts were adult viable, indicating FA genes in zebrafish are generally not essential for early development. None of the mutant fish displayed gross developmental abnormalities except for fancp-/- fish, which were significantly smaller in length than their wildtype clutch mates. Complete female-to-male sex reversal was observed in knockouts for 12/17 FA genes, while partial sex reversal was seen for the other five gene knockouts. All adult females were fertile, and among the adult males, all were fertile except for the fancd1 mutants and one of the fancj mutants. We report here generation and characterization of zebrafish knockout mutants for 17 FA disease-causing genes, providing an integral resource for understanding the pathophysiology associated with the disrupted FA pathway.

Cracking the Monoubiquitin Code of Genetic Diseases

“…FA is characterized by abnormal development, bone marrow failure, hypogonadism, and marked cancer susceptibility. Autosomal recessive mutations in any one of 20 genes (FANCA to FANCQ) result in this genetic disorder, and collectively, the FANC gene products function in a FA−DNA crosslink repair pathway [99]. Several of the FANC genes form a large monoubiquitination complex (FA core complex).…”

“…Several animal models were generated to study the disease. Ube2t knockout in zebrafish leads to hypersensitivity to DNA damage and reversion of female-to-male sex [99], reflecting the hypogonadism phenotype occurring in FA patients. Fancl knockout in mice leads to decrease of fertility and defects in the proliferation of germ cells.…”

A subset of SMN complex members have a specific role in tissue regeneration via ERBB pathway-mediated proliferation

“…7b, data not shown). For the smn1 mutation, the tp53 and smn1 genes in zebrafish were both on chromosome 5, so we obtained double mutants carrying homozygous mutations for an smn1 2 bp insertion and a tp53 7 bp deletion, by injecting smn1 CRISPR guide RNAs into embryos harboring a homozygous 7 bp tp53 deletion mutation 34 and raised those fish for inbreeding. Consistent with the results of the gemin5/tp53 double mutant, tp53 mutants did not rescue the regeneration deficiency or the adult survival of smn1 mutants (data not shown).…”

Zebrafish Photoreceptor Degeneration and Regeneration Research to Understand Hereditary Human Blindness

“…Reverse genetic screening techniques have been used with invertebrate animal models and cell culture systems to identify genes and pathways involved in various biological processes; however, their use with in vivo vertebrate model systems has been challenging [56]. Recently, several zebrafish labs have shown the feasibility of CRISPR/Cas-based mutagenesis assays to isolate high numbers of mutants focused on synapsis [56], thyroid morphogenesis and function [57], and the Fanconi Anemia pathway, which is involved in genomic instability syndrome, resulting in aplastic anemia [58]. One study screened 54 ciliary genes and isolated 8 mutants that were required for retinal development [59].…”