Adult-onset inflammatory syndromes often manifest with overlapping clinical features. Variants in ubiquitin-related genes, previously implicated in autoinflammatory disease, may define new disorders.

Factors contributing to retroviral integration have been intractable because past studies have not precisely located genomic sites of proviruses in sufficient numbers for significant analysis. In this study, 903 murine leukemia virus (MLV) and 379 human immunodeficiency virus–1 (HIV-1) integrations in the human genome were mapped. The data showed that MLV preferred integration near the start of transcriptional units (either upstream or downstream) whereas HIV-1 preferred integration anywhere in the transcriptional unit but not upstream of the transcriptional start. Defining different integration site preferences for retroviruses will have important ramifications for gene therapy and may aid in our understanding of the factors directing the integration process.

The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis. Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F₁ generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/Cas9 is sixfold more efficient than other techniques. We show that the majority of published “rules” for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5′ end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies.

It is estimated that ∼2500 genes are essential for the normal development of a zebrafish embryo. A mutation in any one of these genes can result in a visible developmental defect, usually followed by the death of the embryo or larva by days 5–7 of age. We are performing a large-scale insertional mutagenesis screen in the zebrafish with the goal of isolating ∼1000 embryonic mutations. We plan to clone a significant fraction of the mutated genes, as these are the genes important for normal embryogenesis of a vertebrate. To achieve this goal, we prepared ∼36,000 founder fish by injecting blastula-stage embryos with one of two pseudotyped retroviruses. We estimate that together these fish harbor between 500,000–1,000,000 proviral insertions in their germ lines. The protocol we have devised and the size of our facility allow us to breed ∼80,000–150,000 of these insertions to homozygosity within 2 years. Because a pilot screen conducted earlier in our laboratory revealed that the frequency of mutations obtained with this type of insertional mutagen is 1 embryonic lethal mutation per 70–100 proviral insertions, screening 100,000 insertions should yield at least 1000 mutants. Here we describe the protocol for the screen and initial results with the first of the two retroviral vectors used, a virus designated F₅. We screened an estimated 760 insertions among F₃ progeny from 92 F₂ families and obtained 9 recessive embryonic lethal mutations. Thus, the efficiency of mutagenesis with this viral vector is approximately one-ninth that observed with the chemical mutagen ENU in zebrafish. We have also obtained two dominant mutations, one of which is described here. As expected, mutated genes can be readily identified. So far, genes mutated in four of the nine recessive mutants and one of the two dominant mutants have been cloned. Further improvements to this technology could make large-scale insertional mutagenesis screening and rapid gene cloning accessible to relatively small zebrafish laboratories.

The evolutionary forces that establish and hone target gene networks of transcription factors are largely unknown. Transposition of retroelements may play a role, but its global importance, beyond a few well described examples for isolated genes, is not clear. We report that LTR class I endogenous retrovirus (ERV) retroelements impact considerably the transcriptional network of human tumor suppressor protein p53. A total of 1,509 of ≈319,000 human ERV LTR regions have a near-perfect p53 DNA binding site. The LTR10 and MER61 families are particularly enriched for copies with a p53 site. These ERV families are primate-specific and transposed actively near the time when the New World and Old World monkey lineages split. Other mammalian species lack these p53 response elements. Analysis of published genomewide ChIP data for p53 indicates that more than one-third of identified p53 binding sites are accounted for by ERV copies with a p53 site. ChIP and expression studies for individual genes indicate that human ERV p53 sites are likely part of the p53 transcriptional program and direct regulation of p53 target genes. These results demonstrate how retroelements can significantly shape the regulatory network of a transcription factor in a species-specific manner.