Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.

https://zenodo.org/record/1230922/files/article.pdf

A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

We describe a DNA sequencing technology in which a commonly available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic DNA sequencing automation. We apply this technology to resequence an evolved strain of Escherichia coli at less than one error per million consensus bases. A cell-free, mate-paired library provided single DNA molecules that were amplified in parallel to 1-micrometer beads by emulsion polymerase chain reaction. Millions of beads were immobilized in a polyacrylamide gel and subjected to automated cycles of sequencing by ligation and four-color imaging. Cost per base was roughly one-ninth as much as that of conventional sequencing. Our protocols were implemented with off-the-shelf instrumentation and reagents.

https://science.sciencemag.org/content/sci/309/5741/1728.full.pdf

Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome

Arabidopsis (rockcress) is a genus in the family Brassicaceae. Representatives of Brassicaceae family are small-flowering plants, some of them
are important crops such as cabbage, cauliflower, radish, and canola.
Arabidopsis genus includes nine species and eight subspecies. The
subspecies delimitation is quite recent, and is based on morphological
and molecular phylogenetics (Elizabeth 2000). Arabidopsis thalianacommonly known as thale cress, has 10 chromosomes in diploid stage
and was the first plant to have its entire genome sequenced. Most of the
species in Arabidopsis are indigenous to Europe and only two species are
found in North America and Asia.

Arabidopsis Genome Initiative

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.

/pdf/a-nomenclature-for-restriction-enzymes-dna-19svz4gt01.pdf

A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes

Biology of DNA restriction.

The type III restriction endonuclease EcoP15I was used in isolating fragments of 26 bp from defined positions of cDNAs. We call this substantially improved variant to the conventional serial analysis of gene expression (SAGE) procedure “SuperSAGE.” By applying SuperSAGE to Magnaporthe grisea (blast)-infected rice leaves, gene expression profiles of both the rice host and blast fungus were simultaneously monitored by making use of the fully sequenced genomes of both organisms, revealing that the hydrophobin gene is the most actively transcribed M. grisea gene in blast-infected rice leaves. Moreover, SuperSAGE was applied to study gene expression changes before the so-called hypersensitive response in INF1 elicitor-treated Nicotiana benthamiana, a “nonmodel” organism for which no DNA database is available. Again, SuperSAGE allowed rapid identification of genes up- or down-regulated by the elicitor. Surprisingly, many of the down-regulated genes coded for proteins involved in photosynthesis. SuperSAGE will be especially useful for transcriptome profiling of two or more interacting organisms like hosts and pathogens, and of organisms, for which no DNA database is available.

/pdf/gene-expression-analysis-of-plant-host-pathogen-interactions-o9mb781fi5.pdf

Gene expression analysis of plant host-pathogen interactions by SuperSAGE

A fluorescence based non-radioactive electrophoretic mobility shift assay.

SuperSAGE: Interaction transcriptomics with SuperSAGE

On the divalent metal ion dependence of DNA cleavage by restriction endonucleases of the EcoRI family.

Orthodox Type IIP restriction endonucleases, which are commonly used in molecular biological work, recognize a single palindromic DNA recognition sequence and cleave within or near this sequence. Several new studies have reported on structural and biochemical peculiarities of restriction endonucleases that differ from the orthodox in that they require two copies of a particular DNA recognition sequence to cleave the DNA. These two sites requiring restriction endonucleases belong to different subtypes of Type II restriction endonucleases, namely Types IIE, IIF and IIS. We compare enzymes of these three types with regard to their DNA recognition and cleavage properties. The simultaneous recognition of two identical DNA sites by these restriction endonucleases ensures that single unmethylated recognition sites do not lead to chromosomal DNA cleavage, and might reflect evolutionary connections to other DNA processing proteins that specifically function with two sites.

/pdf/diversity-of-type-ii-restriction-endonucleases-that-require-3sk74vk9ak.pdf

Monika Reuter

Papers

Gene expression analysis of plant host-pathogen interactions by SuperSAGE

A fluorescence based non-radioactive electrophoretic mobility shift assay.

SuperSAGE: Interaction transcriptomics with SuperSAGE

On the divalent metal ion dependence of DNA cleavage by restriction endonucleases of the EcoRI family.

Diversity of type II restriction endonucleases that require two DNA recognition sites.