Genome

About: Genome is a research topic. Over the lifetime, 74231 publications have been published within this topic receiving 3819713 citations.

CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.

Abstract: Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a transcriptional activation-based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator-like (TALs) effectors. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1-3 and 1-2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.

The Diploid Genome Sequence of an Individual Human

Abstract: Presented here is a genome sequence of an individual human. It was produced from ∼32 million random DNA fragments, sequenced by Sanger dideoxy technology and assembled into 4,528 scaffolds, comprising 2,810 million bases (Mb) of contiguous sequence with approximately 7.5-fold coverage for any given region. We developed a modified version of the Celera assembler to facilitate the identification and comparison of alternate alleles within this individual diploid genome. Comparison of this genome and the National Center for Biotechnology Information human reference assembly revealed more than 4.1 million DNA variants, encompassing 12.3 Mb. These variants (of which 1,288,319 were novel) included 3,213,401 single nucleotide polymorphisms (SNPs), 53,823 block substitutions (2–206 bp), 292,102 heterozygous insertion/deletion events (indels)(1–571 bp), 559,473 homozygous indels (1–82,711 bp), 90 inversions, as well as numerous segmental duplications and copy number variation regions. Non-SNP DNA variation accounts for 22% of all events identified in the donor, however they involve 74% of all variant bases. This suggests an important role for non-SNP genetic alterations in defining the diploid genome structure. Moreover, 44% of genes were heterozygous for one or more variants. Using a novel haplotype assembly strategy, we were able to span 1.5 Gb of genome sequence in segments >200 kb, providing further precision to the diploid nature of the genome. These data depict a definitive molecular portrait of a diploid human genome that provides a starting point for future genome comparisons and enables an era of individualized genomic information.

Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: the tortoise and the hare III

Abstract: Although the chloroplast genome contains many noncoding regions, relatively few have been exploited for interspecific phylogenetic and intraspecific phylogeographic studies. In our recent evaluation of the phylogenetic utility of 21 noncoding chloroplast regions, we found the most widely used noncoding regions are among the least variable, but the more variable regions have rarely been employed. That study led us to conclude that there may be unexplored regions of the chloroplast genome that have even higher relative levels of variability. To explore the potential variability of previously unexplored regions, we compared three pairs of single-copy chloroplast genome sequences in three disparate angiosperm lineages: Atropa vs. Nicotiana (asterids); Lotus vs. Medicago (rosids); and Saccharum vs. Oryza (monocots). These three separate sequence alignments highlighted 13 mutational hotspots that may be more variable than the best regions of our former study. These 13 regions were then selected for a more detailed analysis. Here we show that nine of these newly explored regions (rpl32-trnL((UAG)), trnQ((UUG))-5'rps16, 3'trnV((UAC))-ndhC, ndhF-rpl32, psbD-trnT((GGU)), psbJ-petA, 3'rps16-5'trnK((UUU)), atpI-atpH, and petL-psbE) offer levels of variation better than the best regions identified in our earlier study and are therefore likely to be the best choices for molecular studies at low taxonomic levels.

Sequencing and comparison of yeast species to identify genes and regulatory elements

Abstract: Identifying the functional elements encoded in a genome is one of the principal challenges in modern biology. Comparative genomics should offer a powerful, general approach. Here, we present a comparative analysis of the yeast Saccharomyces cerevisiae based on high-quality draft sequences of three related species (S. paradoxus, S. mikatae and S. bayanus). We first aligned the genomes and characterized their evolution, defining the regions and mechanisms of change. We then developed methods for direct identification of genes and regulatory motifs. The gene analysis yielded a major revision to the yeast gene catalogue, affecting approximately 15% of all genes and reducing the total count by about 500 genes. The motif analysis automatically identified 72 genome-wide elements, including most known regulatory motifs and numerous new motifs. We inferred a putative function for most of these motifs, and provided insights into their combinatorial interactions. The results have implications for genome analysis of diverse organisms, including the human.

In vivo genome editing using Staphylococcus aureus Cas9

Abstract: The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.