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  • Gary Ward2014 Nov 04 9:05 p.m. (yesterday) 2 of 2 people found this helpful

    This is a beautiful and clear demonstration of how Toxoplasma gondii can serve as both a useful model organism for the study of other apicomplexan parasites, and powerful surrogate system for small molecule screening. By complementing TgCDPK3 with Plasmodium falciparum CDPK1 (PfCDPK1), the group was able to confirm the functional localization dependence of PfCDPK1 and identify compounds that inhibit both PfCDPK1 and TgCDPK3, as well as those that inhibit PfCDPK1 alone. This work and the work of Sharling et al. PLOS Negl Trop Dis [2010] 4: e794 and others provide good examples of how studying T. gondii may be useful to understanding other apicomplexan parasites from a drug development standpoint.

    Posted by Gary Ward on behalf of the University of Vermont Toxoplasma Journal Club (UVM ToxoJC); members include Sam Ashley, Jenna Foderaro, Anne Kelsen, Shruthi Krishnamurthy, Jacqueline Leung, Pramod Rompikuntal & Gary Ward

  • Fillip Port2014 Nov 05 06:09 a.m. (yesterday) 4 of 4 people found this helpful

    This paper reports on the use of the H1 promoter to drive expression of gRNAs for CRISPR/Cas genome engineering. The authors demonstrate that gRNAs expressed from H1 can efficiently modify the genome of cultured human cells in conjunction with Cas9 endonuclease. Interestingly, gRNA expression levels from H1 are lower than from the commonly used U6 promoter. Although this can negatively effect mutagenesis rates at the on-target site, it can also increase CRISPR/Cas specificity, as high activity is more likely to lead to off-target effects. This makes the H1 promoter a potentially useful tool for CRISPR/Cas genome engineering in human cells.

    However, the authors suggest that their results have much more general significance by expanding the CRISPR/Cas genome targeting space. This is based on the assumption that the U6 promoter requires a G initiation nucleotide (but see comment below and reference therein), which according to the authors constrains genomic target sites to GN19NGG. This assertion is surprising as it is common practice in the CRISPR field to target sites that do not start with a G by simply adding a (often mismatched) G to the corresponding gRNA or to replace the first nucleotide with a G to create a gRNA that is mismatched at the first position. The authors acknowledge these strategies in the first paragraph of their discussion, but cite six papers as providing evidence that 5’ extensions or truncations reduce gRNA efficiency. However, these papers in fact provide evidence that 5’ extensions or truncations of a single nucleotide often have no effect on activity and when they do the effect is usually minor (modified gRNAs usually retain >80% activity). Furthermore, the authors fail to cite another study that shows that small gRNA truncations often retain full activity but have reduced off-target effects (Fu Y, 2014). Therefore, much of the published evidence suggests that extending or truncating gRNAs by a single nucleotide has minimal or no effects on activity. As a result it is in principle possible to target any genomic site adjacent to a PAM motif with gRNAs expressed from a U6 promoter and hence the H1 promoter, although potentially useful, does not expand the CRISPR target space.

  • Dan Laks2014 Nov 05 10:01 a.m. (yesterday) 1 of 1 people found this helpful

    These data support earlier findings that chronic Hg exposure results in depletion of LH:

    http://www.ncbi.nlm.nih.gov/pubmed/19914008 http://www.ncbi.nlm.nih.gov/pubmed/19697139

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