Modulating CRISPR gene drive activity through nucleocytoplasmic localization of Cas9 in S. cerevisiae

ABSTRACTThe bacterial CRISPR/Cas genome editing system has provided a major breakthrough in molecular biology. One use of this technology is within a nuclease-based gene drive. This type of system can install a genetic element within a population at unnatural rates. Combatting of vector-borne diseases carried by metazoans could benefit from a delivery system that bypasses traditional Mendelian laws of segregation. Recently, laboratory studies in fungi, insects, and even mice, have demonstrated successful propagation of CRISPR gene drives and the potential utility of this type of mechanism. However, current gene drives still face challenges including evolved resistance, containment, and the consequences of application in wild populations. In this study, we use an artificial gene drive system in budding yeast to explore mechanisms to modulate nuclease activity of Cas9 through its nucleocytoplasmic localization. We examine non-native nuclear localization sequences on Cas9 fusion proteins in vivo and demonstrate that appended signals can titrate gene drive activity and serve as a potential molecular safeguard.

Cootie Genetics

“Cootie Genetics” is a hands-on, inquiry-based activity that enables students to learn the Mendelian laws of inheritance and gain an understanding of genetics principles and terminology. The activity begins with two true-breeding Cooties of the same species that exhibit five observable trait differences. Students observe the retention or loss of traits among first-generation heterozygotes, hypothesize what happened to these traits, and design an experiment to test their hypotheses by mating the first-generation Cooties. With the second generation, Mendel’s principles of segregation and independent assortment of alleles are observed; dominant and recessive traits and tools students need to construct Punnett squares are apparent.

The novel Arabidopsis thaliana svt2 suppressor of the ascorbic acid-deficient mutant vtc1-1 exhibits phenotypic and genotypic instability

Ascorbic acid is a potent antioxidant that detoxifies reactive oxygen species when plants are exposed to unfavorable environmental conditions. In addition to its antioxidant properties, ascorbic acid and its biosynthetic precursors fulfill a variety of other physiological and molecular functions. A mutation in the ascorbic acid biosynthesis gene VTC1, which encodes GDP-mannose pyrophosphorylase, results in conditional root growth inhibition in the presence of ammonium. To isolate suppressors of vtc1-1, which is in the Arabidopsis Columbia-0 background, seeds of the mutant were subjected to ethyl methanesulfonate mutagenesis. A suppressor mutant of vtc1-1 2, svt2, with wild-type levels of ascorbic acid and root growth similar to the wild type in the presence of ammonium was isolated. Interestingly, svt2 has Arabidopsis Landsberg erecta features, although svt2 is delayed in flowering and has an enlarged morphology. Moreover, the svt2 genotype shares similarities with Ler polymorphism markers and sequences, despite the fact that the mutant derived from mutagenesis of Col-0 vtc1-1 seed. We provide evidence that svt2 is not an artifact of the experiment, a contamination of Ler seed, or a result of outcrossing of the svt2 mutant with Ler pollen. Instead, our results show that svt2 exhibits transgenerational genotypic and phenotypic instability, which is manifested in a fraction of svt2 progeny, producing revertants that have Col-like phenotypic and genotypic characteristics. Some of those Col-like revertants then revert back to svt2-like plants in the subsequent generation. Our findings have important implications for undiscovered phenomena in transmitting genetic information in addition to the Mendelian laws of inheritance. Our results suggest that stress can trigger a genome restoration mechanism that could be advantageous for plants to survive environmental changes for which the ancestral genes were better adapted.

A study of biochemical polymorphism in carp (Cyprinus Carpio): Detect new alleles in transferrin

This study was carried out at the college of agriculture and marine science centre, Basrah university. The present study was conducted to investigate the existence of polymorphism at transferrin (Tf) locus in the Carp (Cyprinus carpio). A polyacrylamide gel electrophoresis (PAGE) under alkaline condition method was used to distinguish Carp Tf alleles. Use the gel documentation program in this study. Analysis of 116 animals revealed that all animals were polymorphic, showing many genotypes. There was very clear biodiversity in the Tf gene. Seven Tf genotypes consisting of 4 homozygote types (CC, DD, FF and GG) and two heterozygote types (CD, DG and FG) were detected. These fractions are controlled by co-dominant autosomal genes according to the Mendelian laws of inheritance. The highest gene frequencies were calculated 0.50 for Tf D, 0.26 for Tf F and 0.12 for C and G. thus, carp (Cyprinus carpio) assemblages consistently tended to be more predominant to D allele. Differences between expected number and observed number for transferrin genotypes were no significant. This is useful in genetic improvement process through the selection. As far as we know, this is the first large-scale analysis on the genetic polymorphism in carp (Cyprinus carpio). Polyacrylamide electrophoresis, the technique employed in this study, allows rapid and efficient screening for the presence of polymorphism in Tf.