CRISPR off-target analysis in genetically engineered rats and mice.

Despite widespread use of CRISPR, comprehensive data on the frequency and impact of Cas9-mediated off-targets in modified rodents are limited. Here we present deep-sequencing data from 81 genome-editing projects on mouse and rat genomes at 1,423 predicted off-target sites, 32 of which were confirmed, and show that high-fidelity Cas9 versions reduced off-target mutation rates in vivo. Using whole-genome sequencing data from ten mouse embryos, treated with a single guide RNA (sgRNA), and from their genetic parents, we found 43 off-targets, 30 of which were predicted by an adapted version of GUIDE-seq.

N-Acylation of Oxazolidinones via Aerobic Oxidative NHC Catalysis.

The first N-acylation of synthetically useful oxazolidinones with aldehydes using aerobic oxidative NHC catalysis is reported. The reaction offers a broad scope of functionalized oxazolidinones in good to excellent yields. Careful choice of electron transfer mediators proved pivotal to achieve efficient aerobic N-acylation, which has previously proven difficult using NHC catalysis. The methodology allows a mild entry to acylated oxazolidinones, avoiding the use of hazardous and reactive prefunctionalized substrates.

Ionic liquids as precatalysts in the highly stereoselective conjugate addition of α,ß-unsaturated aldehydes to chalcones.

Imidazolium-based ionic liquids (ILs) serve both as recyclable reaction media and as precatalysts for the N-heterocyclic carbene-catalyzed conjugate addition of α,ß-unsaturated aldehydes to chalcones. The reaction produces a broad scope of 1,6-ketoesters incorporating an anti-diphenyl moiety in high yields and with high stereoselectivity. In recycling experiments, the IL can be reused up to five times with retained reactivity and selectivity. Moreover, the 1,6-ketoesters form self-assembled organogels in aliphatic hydrocarbons. The reaction protocol is robust, easily operated, scalable and highly functionalized compounds can be obtained from inexpensive and readily accessible starting materials.

Examination of FGFRL1 as a candidate gene for diaphragmatic defects at chromosome 4p16.3 shows that Fgfrl1 null mice have reduced expression of Tpm3, sarcomere genes and Lrtm1 in the diaphragm.

Fgfrl1 (also known as Fgfr5; OMIM 605830) homozygous null mice have thin, amuscular diaphragms and die at birth because of diaphragm hypoplasia. FGFRL1 is located at 4p16.3, and this chromosome region can be deleted in patients with congenital diaphragmatic hernia (CDH). We examined FGFRL1 as a candidate gene for the diaphragmatic defects associated with 4p16.3 deletions and re-sequenced this gene in 54 patients with CDH. We confirmed six known coding single nucleotide polymorphisms (SNPs): c.209G > A (p.Pro20Pro), c.977G > A (p.Pro276Pro), c.1040T > C (p.Asp297Asp), c.1234C > A (p.Pro362Gln), c.1420G > T (p.Arg424Leu), and c.1540C > T (p.Pro464Leu), but we did not identify any gene mutations. We genotyped additional CDH patients for four of these six SNPs, including the three non-synonymous SNPs, to make a total of 200 chromosomes, and found that the allele frequency for the four SNPs, did not differ significantly between patients and normal controls (p > or = 0.05). We then used Affymetrix Genechip Mouse Gene 1.0 ST arrays and found eight genes with significantly reduced expression levels in the diaphragms of Fgfrl1 homozygous null mice when compared with wildtype mice-Tpm3, Fgfrl1 (p = 0.004), Myl2, Lrtm1, Myh4, Myl3, Myh7 and Hephl1. Lrtm1 is closely related to Slit3, a protein associated with herniation of the central tendon of the diaphragm in mice. The Slit proteins are known to regulate axon branching and cell migration, and inhibition of Slit3 reduces cell motility and decreases the expression of Rac and Cdc42, two genes that are essential for myoblast fusion. Further studies to determine if Lrtm1 has a similar function to Slit3 and if reduced Fgfrl1 expression can cause diaphragm hypoplasia through a mechanism involving decreased myoblast motility and/or myoblast fusion, seem indicated.

Alternative splicing in the differentiation of human embryonic stem cells into cardiac precursors.

The role of alternative splicing in self-renewal, pluripotency and tissue lineage specification of human embryonic stem cells (hESCs) is largely unknown. To better define these regulatory cues, we modified the H9 hESC line to allow selection of pluripotent hESCs by neomycin resistance and cardiac progenitors by puromycin resistance. Exon-level microarray expression data from undifferentiated hESCs and cardiac and neural precursors were used to identify splice isoforms with cardiac-restricted or common cardiac/neural differentiation expression patterns. Splice events for these groups corresponded to the pathways of cytoskeletal remodeling, RNA splicing, muscle specification, and cell cycle checkpoint control as well as genes with serine/threonine kinase and helicase activity. Using a new program named AltAnalyze (http://www.AltAnalyze.org), we identified novel changes in protein domain and microRNA binding site architecture that were predicted to affect protein function and expression. These included an enrichment of splice isoforms that oppose cell-cycle arrest in hESCs and that promote calcium signaling and cardiac development in cardiac precursors. By combining genome-wide predictions of alternative splicing with new functional annotations, our data suggest potential mechanisms that may influence lineage commitment and hESC maintenance at the level of specific splice isoforms and microRNA regulation.

Oxidative organocatalytic chemoselective N-acylation of heterocycles with aromatic and conjugated aldehydes.

Selective acylation of indoles is cumbersome often involving the need for sensitive and reactive acyl chloride derivatives or coupling reagents. Here we report a mild, functional group tolerant and highly chemoselective oxidative carbene catalyzed N-acylation of indoles with aldehydes. The acylation has a broad substrate scope and is compatible with substituents on both the aldehyde and the indole reaction partner. Furthermore, aza-heterocycles such as pyrrole and indazole can also be used as nucleophiles in this reaction providing the corresponding amide congeners in good yield.

Highly Stereoselective Synthesis of 1,6-Ketoesters Mediated by Ionic Liquids: A Three-component Reaction Enabling Rapid Access to a New Class of Low Molecular Weight Gelators.

In organic chemistry ionic liquids (ILs) have emerged as safe and recyclable reaction solvents. In the presence of a base ILs can be deprotonated to form catalytically active N-Heterocyclic Carbenes (NHCs). Here we have used ILs as precatalysts in the addition of α,ß-unsaturated aldehydes to chalcones to form 1,6-ketoesters, incorporating an anti-diphenyl moiety in a highly stereoselective fashion. The reaction has a broad substrate scope and several functional groups and heteroaromatics can be integrated into the ketoester backbone in generally good yields with maintained stereoselectivity. The reaction protocol is robust and scalable. The starting materials are inexpensive and the products can be obtained after simple filtration, avoiding solvent-demanding chromatography. Furthermore, the IL can be recycled up to 5 times without any loss of reactivity. Moreover, the 1,6-ketoester end product is a potent gelator in several hydrocarbon based solvents. The method enables rapid access to and evaluation of a new class of low molecular weight gelators (LMWGs) from recyclable and inexpensive starting materials.

Conserved transcriptional unit organization of the cag pathogenicity island among Helicobacter pylori strains.

The Helicobacter pyloricag pathogenicity island (cag PAI) encodes a type IV secretion system that is more commonly found in strains isolated from patients with gastroduodenal disease than from those with asymptomatic gastritis. Genome-wide organization of the transcriptional units in H. pylori strain 26695 was recently established using RNA sequence analysis (Sharma et al., 2010). Here we used quantitative reverse-transcription polymerase chain reaction of open reading frames and intergenic regions to identify putative cag PAI operons in H. pylori; these operons were analyzed further by transcript profiling after deletion of selected promoter regions. Additionally, we used a promoter-trap system to identify functional cag PAI promoters. The results demonstrated that expression of genes on the H. pyloricag PAI varies by nearly five orders of magnitude and that the organization of cag PAI genes into transcriptional units is conserved among several H. pylori strains, including, 26695, J99, G27, and J166. We found evidence for 20 transcripts within the cag PAI, many of which likely overlap. Our data suggests that there are at least 11 operons: cag1-4, cag3-4, cag10-9, cag8-7, cag6-5, cag11-12, cag16-17, cag19-18, cag21-20, cag23-22, and cag25-24, as well as five monocistronic genes (cag4, cag13, cag14, cag15, and cag26). Additionally, the location of four of our functionally identified promoters suggests they are directing expression of, in one case, a truncated version of cag26 and in the other three, transcripts that are antisense to cag7, cag17, and cag23. We verified expression of two of these antisense transcripts, those antisense to cag17 and cag23, by reverse-transcription polymerase chain reaction. Taken together, our results suggest that the cag PAI transcriptional profile is generally conserved among H. pylori strains, 26695, J99, G27, and J166, and is likely complex.