Positive Selection in the Evolution of Mammalian CRISPs.

Cysteine-RIch Secretory Proteins (CRISPs) constitute a versatile family, with functions in reptilian venom and mammalian reproduction. Mammals generally express three CRISPs, four in mice, and all are highly expressed in male reproductive tissues, either testis or accessory organs. Because reproductive proteins often evolve adaptively in response to post-copulatory sexual selection, we hypothesized that mammalian CRISPs, with important roles in male reproduction, could have undergone positive selection promoting their divergence. We explored the molecular adaptation of mammalian CRISPs applying phylogenetic methods. Our analyses revealed the evidence of positive selection in all mammalian CRISPs. The intensity of positive selection was heterogeneous among CRISP members, being stronger in CRISP3 than in CRISP1 and CRISP2, and also across functional domains, having stronger impact on Pathogenesis-Related 1 (PR-1) in CRISP2 and on Ion Channel Regulator (ICR) in CRISP1 and CRISP3. In addition, we discovered a new CRISP in some rodent species, suggesting that the acquisition of new CRISP components could contribute to male reproductive success or to acquire new physiological roles. Signatures of positive selection were not focused on any particular mammalian group, suggesting that adaptive evolution is a recurrent pattern in mammalian CRISPs. Our findings support a model of CRISP family diversification driven by episodes of duplication and posterior neofunctionalization, and provide potential adaptive changes responsible for interspecific differences in CRISPs activity.

Genome Editing by CRISPR/Cas9: A Game Change in the Genetic Manipulation of Protists.

Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system has been transformative in biology. Originally discovered as an adaptive prokaryotic immune system, CRISPR/Cas9 has been repurposed for genome editing in a broad range of model organisms, from yeast to mammalian cells. Protist parasites are unicellular organisms producing important human diseases that affect millions of people around the world. For many of these diseases, such as malaria, Chagas disease, leishmaniasis and cryptosporidiosis, there are no effective treatments or vaccines available. The recent adaptation of the CRISPR/Cas9 technology to several protist models will be playing a key role in the functional study of their proteins, in the characterization of their metabolic pathways, and in the understanding of their biology, and will facilitate the search for new chemotherapeutic targets. In this work we review recent studies where the CRISPR/Cas9 system was adapted to protist parasites, particularly to Apicomplexans and trypanosomatids, emphasizing the different molecular strategies used for genome editing of each organism, as well as their advantages. We also discuss the potential usefulness of this technology in the green alga Chlamydomonas reinhardtii.

In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by CRISPR/Cas9.

Deregulated expression of high-risk human papillomavirus oncogenes (E6 and E7) is a pivotal event for pathogenesis and progression in cervical cancer. Both viral oncogenes are therefore regarded as ideal therapeutic targets. In the hope of developing a gene-specific therapy for HPV-related cancer, we established CRISPR/Cas9 targeting promoter of HPV 16 E6/E7 and targeting E6, E7 transcript, transduced the CRISPR/Cas9 into cervical HPV-16-positive cell line SiHa. The results showed that CRISPR/Cas9 targeting promoter, as well as targeting E6 and E7 resulted in accumulation of p53 and p21 protein, and consequently remarkably reduced the abilities of proliferation of cervical cancer cells in vitro. Then we inoculated subcutaneously cells into nude mice to establish the transplanted tumor animal models, and found dramatically inhibited tumorigenesis and growth of mice incubated by cells with CRISPR/Cas9 targeting (promoter+E6+E7)-transcript. Our results may provide evidence for application of CRISPR/Cas9 targeting HR-HPV key oncogenes, as a new treatment strategy, in cervical and other HPV-associated cancer therapy.