The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.

SPAdes, a new genome assembly algorithm and its applications to single-cell sequencing ( 7th Annual SFAF Meeting, 2012)

Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the power of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.

http://stacks.cdc.gov/view/cdc/29957/cdc_29957_DS1.pdf

CRISPR-Cas systems for editing, regulating and targeting genomes

The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.

/pdf/programmable-base-editing-of-a-t-to-g-c-in-genomic-dna-rn1bxgz6yb.pdf

Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage

An annotated reference sequence representing the hexaploid bread wheat genome in 21 pseudomolecules has been analyzed to identify the distribution and genomic context of coding and noncoding elements across the A, B, and D subgenomes. With an estimated coverage of 94% of the genome and containing 107,891 high-confidence gene models, this assembly enabled the discovery of tissue- and developmental stage-related coexpression networks by providing a transcriptome atlas representing major stages of wheat development. Dynamics of complex gene families involved in environmental adaptation and end-use quality were revealed at subgenome resolution and contextualized to known agronomic single-gene or quantitative trait loci. This community resource establishes the foundation for accelerating wheat research and application through improved understanding of wheat biology and genomics-assisted breeding.

Shifting the limits in wheat research and breeding using a fully annotated reference genome

A recently developed adenine base editor (ABE) efficiently converts A to G and is potentially useful for clinical applications. However, its precision and efficiency in vivo remains to be addressed. Here we achieve A-to-G conversion in vivo at frequencies up to 100% by microinjection of ABE mRNA together with sgRNAs. We then generate mouse models harboring clinically relevant mutations at Ar and Hoxd13, which recapitulates respective clinical defects. Furthermore, we achieve both C-to-T and A-to-G base editing by using a combination of ABE and SaBE3, thus creating mouse model harboring multiple mutations. We also demonstrate the specificity of ABE by deep sequencing and whole-genome sequencing (WGS). Taken together, ABE is highly efficient and precise in vivo, making it feasible to model and potentially cure relevant genetic diseases. CRISPR-based base editors allow for single nucleotide genome editing in a range of organisms. Here the authors demonstrate the in vivo generation of mouse models carrying clinically relevant mutations using C→T and A→G editors.

/pdf/efficient-generation-of-mouse-models-of-human-diseases-via-p9gafrxed2.pdf

Efficient generation of mouse models of human diseases via ABE- and BE-mediated base editing

Wheat stem (or black) rust, caused by Puccinia graminis f. sp. tritici (Pgt), has been historically among the most devastating global fungal diseases of wheat. The recent occurrence and spread of new virulent races such as Ug99 have prompted global efforts to identify and isolate more effective stem rust resistance (Sr) genes. Here, we report the map-based cloning of the Ug99-effective SrTm5 gene from diploid wheat Triticum monococcum accession PI 306540 that encodes a typical coiled-coil nucleotide-binding leucine-rich repeat protein. This gene, designated as Sr22b, is a new allele of Sr22 with a rare insertion of a large (13.8-kb) retrotransposon into its second intron. Biolistic transformation of an ˜112-kb circular bacterial artificial chromosome plasmid carrying Sr22b into the susceptible wheat variety Fielder was sufficient to confer resistance to stem rust. In a survey of 168 wheat genotypes, Sr22b was present only in cultivated T. monococcum subsp. monococcum accessions but absent in all tested tetraploid and hexaploid wheat lines. We developed a diagnostic molecular marker for Sr22b and successfully introgressed a T. monococcum chromosome segment containing this gene into hexaploid wheat to accelerate its deployment and pyramiding with other Sr genes in wheat breeding programmes. Sr22b can be a valuable component of gene pyramids or transgenic cassettes combining different resistance genes to control this devastating disease.

Identification and characterization of Sr22b, a new allele of the wheat stem rust resistance gene Sr22 effective against the Ug99 race group.

/pdf/genome-edited-crops-how-to-move-them-from-laboratory-to-34nxv5yngs.pdf

Genome-edited crops: how to move them from laboratory to market

The dielectric properties of fresh and aged Ba(Zr0.25Ti0.75)O3 thin films have been investigated. It is found that the aged sample shows a significant decrease in the dielectric constant compared to the fresh one. Furthermore, the aged sample also exhibits abnormal double-peaks-shape butterfly C–V characteristics, which indicates the defect-dipole stabilized domain and/or domain-wall motions. Meanwhile, we found that double-peaks-shape butterfly C–V characteristics become weak and even disappear with increasing of applied electric field and temperature. The present results are discussed in light of the symmetry-conforming principle of point defects.

Aging-induced abnormality of dielectric response under dc bias in Ba(Zr, Ti)O3 thin films

The CRISPR/Cas9 Genome Editing Revolution.

Meadow fescue (Festuca pratensis Huds.) is an important cool-season forage grass in Europe and Asia. We developed a protocol for producing meadow fescue transgenic plants mediated by Agrobacterium tumefaciens transformation. Embryogenic calli derived from mature embryos were transformed with A. tumefaciens strain AGL1 carrying the binary vector pDM805, coding for the phosphinothricin acetyltransferase (bar) and β-glucuronidase (uidA) genes. Bialaphos was used as the selective agent throughout all phases of tissue culture. In total, 40 independent transgenic plants were recovered from 45 bialaphos-resistant callus lines and an average transformation efficiency of 2% was achieved. The time frame from infection of embryogenic calli with Agrobacterium to transferring the transgenic plants to the greenhouse was 18 weeks. In a study of 11 BASTA-resistant transgenic lines, the uidA gene was expressed in 82% of the transgenic lines. Southern blot analysis revealed that 82% of the tested lines integrated one or two copies of the uidA gene.

Caixia Gao

Papers

Identification and characterization of Sr22b, a new allele of the wheat stem rust resistance gene Sr22 effective against the Ug99 race group.

Genome-edited crops: how to move them from laboratory to market

Aging-induced abnormality of dielectric response under dc bias in Ba(Zr, Ti)O3 thin films

The CRISPR/Cas9 Genome Editing Revolution.

Agrobacterium-mediated transformation of meadow fescue (Festuca pratensis Huds.).