Palladium-Catalyzed Saegusa–Ito Oxidation: Synthesis of α,β-Unsaturated Carbonyl Compounds from Trimethylsilyl Enol Ethers

Palladium-catalyzed Saegusa–Ito oxidation of trimethylsilyl enol ethers is possible using Oxone as a stoichiometric oxidant and sodium hydrogen phosphate as a buffer. Cyclic and acyclic enones as well as α,β-unsaturated aldehydes are obtained in good to excellent yields

Copper-Catalyzed Petasis-Type Reaction: A General Route to α-Substituted Amides From Imines, Acid Chlorides, and Organoboron Reagents

A copper-catalyzed Petasis-type reaction of imines, acid chlorides, and organoboranes to form α-substituted amides is described. This reaction does not require the use of activated imines or the transfer of special units from the organoboranes and represent a useful generalization of the Petasis reaction

Heritable Multiplex Genetic Engineering in Rats Using CRISPR/Cas9

<div><p>The CRISPR/Cas9 system has been proven to be an efficient gene-editing tool for genome modification of cells and organisms. Multiplex genetic engineering in rat holds a bright future for the study of complex disease. Here, we show that this system enables the simultaneous disruption of four genes (<i>ApoE</i>, <i>B2m</i>, <i>Prf1</i>, and <i>Prkdc</i>) in rats in one-step, by co-injection of Cas9 mRNA and sgRNAs into fertilized eggs. We further observed the gene modifications are germline transmittable, and confirmed the off-target mutagenesis and mosaicism are rarely detected by comprehensive analysis. Thus, the CRISPR/Cas9 system makes it possible to efficiently and reliably generate gene knock-out rats.</p></div

Summary of the mutation of the founder rats.

<p>Single sgRNA/Gene: A mixture of 4 sgRNAs, each targeting a single site in each of the 4 genes. Dual sgRNAs/Gene: Another distinct sgRNA of each gene, together with the tested 4 sgRNAs targeting double site in each of the 4 genes.</p

Cas9:sgRNA-mediated modifications in 4 genes by a mixture of dual sgRNAs for each gene.

<p>(a) PCR identification of sgRNA:Cas9-mediated site-specific cleavage of the endogenous <i>ApoE</i>, <i>B2m</i>, <i>Prf1</i>, and <i>Prkdc</i> loci. The genetic modification analysis was performed by PCR amplification of the targeted fragment in the <i>ApoE</i>, <i>B2m</i>, <i>Prf1</i>, and <i>Prkdc</i> in 25 founder rats (#16∼40) derived from co-microinjection of a mixture of dual sgRNAs for each genes as described in Table S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089413#pone.0089413.s001" target="_blank">File S1</a>. Primer used for PCR amplication was described in Table S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089413#pone.0089413.s001" target="_blank">File S1</a>. Additionally, founder #36, #38 had a larger deletion in the <i>ApoE</i>, the primer <i>ApoE</i>-NS2 and <i>ApoE</i>-NAS2 used for amplification. Founder #38 had a larger deletion in the <i>B2m</i>, the primer <i>B2m</i>-S2 and <i>B2m</i>-AS2 used for amplification. (b) Detection of Cas9:sgRNA-mediated site-specific cleavage of the endogenous <i>ApoE</i>, <i>B2m</i>, <i>Prf1</i>, and <i>Prkdc</i> by T7EN1 cleavage assay. PCR products from (a) were subjected to T7EN1 cleavage assay as described in material and methods.</p

Schematic diagrams of sgRNAs and DNA sequences of targeting genomic loci.

<p>PCR amplicon of the targeted fragment in the <i>ApoE</i>, <i>B2m</i>, <i>Prf1</i>, and <i>Prkdc</i> in all founder rats (#1∼40) were sequenced. The PAM sequence is underlined and highlighted in green; the targeting site are red; the mutations are blue, lower case; insertions (+), deletions (−) or mutant (m) are shown to the right of each allele. N/N indicates positive colonies out of total sequenced. (a) <i>ApoE</i> locus. (b) <i>B2m</i> locus. (c) <i>Prf1</i> locus. (d) <i>Prkdc</i> locus.</p

Analysis of the transmission of the off-target mutation.

<p>(a) Detection of Cas9:sgRNA-mediated off-target cleavage <i>of Prkdc</i> OTS-4 in all founders (#1–40) by T7EN1 cleavage assay. PCR amplicon of <i>Prkdc</i> OTS-4 in all 40 founder rats were subjected to T7EN1 cleavage assay as described in methods. Total 23 founders (*) displayed cleavage bands. (b) PCR products with cleavage bands were cloned and sequenced. Sequence result showed OTS-4 indeed mutagenized in the 23 founders. Indels were also detected around 270 bp downstream of the OTS-4 in most colonies, which may be introduced by PCR amplification when Taq encountering repeat sequence. (c) Detection of Cas9:sgRNA-mediated off-target cleavage of <i>Prkdc</i> OTS-4 in 8 F1 pups derived from founder #3 by T7EN1 cleavage assay. Mutations were detected in 2 F1 pups (4 and 8). (d) DNA sequences of <i>Prkdc</i> OTS-4 in F1 pups 4 and 8. PCR amplicon of the <i>Prkdc</i> OTS-4 in founder #3-derived F1 pups 4 and 8 were sequenced. Sequencing result showed one kind of off-target mutation same as the founder #3 was detected in the offspring, indicating that off-target mutation induced by Cas9:sgRNA was heritable.</p

Generation of multiplex genetic modified rats using CRISPR/Cas9 system.

<p>(a) Detection of Cas9:sgRNA-mediated site-specific cleavage of the endogenous <i>ApoE</i>, <i>B2m</i>, <i>Prf1</i>, and <i>Prkdc</i> by T7EN1 cleavage assay. PCR amplicon of the targeted fragment at the <i>ApoE</i>, <i>B2m</i>, <i>Prf1</i>, and <i>Prkdc</i> in 15 founder rats (#1∼15) were subjected to T7EN1 cleavage assay. Founder #13, which is quadruple gene mutant, was marked with asterisks. (b) DNA sequences of four loci in founder #13. PCR amplicon with cleaved bands in T7EN1 cleavage assay were cloned and sequenced. The PAM sequence was underlined and highlighted in green; the targeting site are red; the mutations are blue, lower case; insertions (+) or deletions (−) are shown to the right of each allele.</p