Browsing by Subject "DNA / chemistry"
Now showing 1 - 2 of 2
- Results Per Page
- Sort Options
Item Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations(Springer Nature, 2016-11-22) Zuo, Zhicheng; Liu, JinThe CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg(2+) ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg(2+) bound. The simulation results showed that binding of two Mg(2+) ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.Item Precision DNA Mixture Interpretation with Single-Cell Profiling(MDPI, 2021-10-20) Ge, Jianye; King, Jonathan L.; Smuts, Amy; Budowle, BruceWet-lab based studies have exploited emerging single-cell technologies to address the challenges of interpreting forensic mixture evidence. However, little effort has been dedicated to developing a systematic approach to interpreting the single-cell profiles derived from the mixtures. This study is the first attempt to develop a comprehensive interpretation workflow in which single-cell profiles from mixtures are interpreted individually and holistically. In this approach, the genotypes from each cell are assessed, the number of contributors (NOC) of the single-cell profiles is estimated, followed by developing a consensus profile of each contributor, and finally the consensus profile(s) can be used for a DNA database search or comparing with known profiles to determine their potential sources. The potential of this single-cell interpretation workflow was assessed by simulation with various mixture scenarios and empirical allele drop-out and drop-in rates, the accuracies of estimating the NOC, the accuracies of recovering the true alleles by consensus, and the capabilities of deconvolving mixtures with related contributors. The results support that the single-cell based mixture interpretation can provide a precision that cannot beachieved with current standard CE-STR analyses. A new paradigm for mixture interpretation is available to enhance the interpretation of forensic genetic casework.