Expression and purification of cell-penetrating Cas9 and Cas12a enzymes for peptide-assisted genome editing.

Recent advances in CRISPR-Cas genomic editors have shifted us ever closer to achieving the ultimate therapeutic goal of accomplishing any edit in any cell. However, delivery of this editing machinery to primary cells with high efficiency while avoiding cellular toxicity remains a formidable challenge. Peptide-Assisted Genome Editing (PAGE) provides a simple, modular, and rapid approach for the protein-based delivery of CRISPR-Cas proteins or ribonucleoprotein complexes into primary cells with high efficiency and minimal cytotoxicity.

MSA: scalable DNA methylation screening BeadChip for high-throughput trait association studies.

The Infinium DNA Methylation BeadChips have significantly contributed to population-scale epigenetics research by enabling epigenome-wide trait association discoveries. Here, we design, describe, and experimentally verify a new iteration of this technology, the Methylation Screening Array (MSA), to focus on human trait screening and discovery. This array utilizes extensive data from previous Infinium platform-based epigenome-wide association studies (EWAS).

Cooperativity between Cas9 and hyperactive AID establishes broad and diversifying mutational footprints in base editors.

The partnership of DNA deaminase enzymes with CRISPR-Cas nucleases is now a well-established method to enable targeted genomic base editing. However, an understanding of how Cas9 and DNA deaminases collaborate to shape base editor (BE) outcomes has been lacking. Here, we support a novel mechanistic model of base editing by deriving a range of hyperactive activation-induced deaminase (AID) base editors (hBEs) and exploiting their characteristic diversifying activity.

Efficient engineering of human and mouse primary cells using peptide-assisted genome editing.

Simple, efficient and well-tolerated delivery of CRISPR genome editing systems into primary cells remains a major challenge. Here we describe an engineered Peptide-Assisted Genome Editing (PAGE) CRISPR-Cas system for rapid and robust editing of primary cells with minimal toxicity. The PAGE system requires only a 30-min incubation with a cell-penetrating Cas9 or Cas12a and a cell-penetrating endosomal escape peptide to achieve robust single and multiplex genome editing.

Action and timing of BacC and BacD in the late stages of biosynthesis of the dipeptide antibiotic bacilysin.

Biosynthesis of the dipeptide antibiotic bacilysin, encoded by the seven Bacillus subtilis genes bacA-G, involves diversion of flux from prephenate to the noncognate amino acid anticapsin. The anticapsin warhead is then ligated to the C-terminus of l-alanine to produce mature bacilysin. We have previously noted the formation of two diastereomers of tetrahydrotyrosine (4S- and 4R-H(4)Tyr) by tandem action of the four purified enzymes BacABGF.

Stereochemical outcome at four stereogenic centers during conversion of prephenate to tetrahydrotyrosine by BacABGF in the bacilysin pathway.

The first four enzymes of the bacilysin antibiotic pathway, BacABGF, convert prephenate to a tetrahydrotyrosine (H(4)Tyr) diastereomer on the way to the anticapsin warhead of the dipeptide antibiotic. BacB takes the BacA product endocyclic-Δ(4),Δ(8)-7R-dihydrohydroxyphenylpyruvate (en-H(2)HPP) and generates a mixture of 3E- and 3Z-olefins of the exocyclic-Δ(3),Δ(5)-dihydrohydroxyphenylpyruvate (ex-H(2)HPP). The NADH-utilizing BacG then catalyzes a conjugate reduction, adding a pro-S hydride equivalent to C(4) to yield tetrahydrohydroxyphenylpyruvate (H(4)HPP), a transamination away (via BacF) from 2S-H(4)Tyr.

Olefin isomerization regiochemistries during tandem action of BacA and BacB on prephenate in bacilysin biosynthesis.

BacA and BacB, the first two enzymes of the bacilysin pathway, convert prephenate to an exocylic regioisomer of dihydrohydroxyphenylpyruvate (ex-H(2)HPP) on the way to the epoxycyclohexanone warhead in the dipeptide antibiotic, bacilysin. BacA decarboxylates prephenate without aromatization, converting the 1,4-diene in prephenate to the endocyclic 1,3-diene in Δ(4),Δ(8)-dihydrohydroxyphenylpyruvate (en-H(2)HPP). BacB then performs an allylic isomerization to bring the diene into conjugation with the 2-ketone in the product Δ(3),Δ(5)-dihydrohydroxyphenylpyruvate (ex-H(2)HPP).

Management of pain with diclofenac after femtosecond-assisted laser in situ keratomileusis.

Purpose: To evaluate the safety and efficacy of topical diclofenac sodium 0.1% after femtosecond laser-assisted laser in situ keratomileusis (LASIK).

Setting: W.

Effect of the thymidylate synthase inhibitors on dUTP and TTP pool levels and the activities of DNA repair glycosylases on uracil and 5-fluorouracil in DNA.

5-Fluorouracil (5-FU), 5-fluorodeoxyuridine (5-dUrd), and raltitrixed (RTX) are anticancer agents that target thymidylate synthase (TS), thereby blocking the conversion of dUMP into dTMP. In budding yeast, 5-FU promotes a large increase in the dUMP/dTMP ratio leading to massive polymerase-catalyzed incorporation of uracil (U) into genomic DNA, and to a lesser extent 5-FU, which are both excised by yeast uracil DNA glycosylase (UNG), leading to DNA fragmentation and cell death. In contrast, the toxicity of 5-FU and RTX in human and mouse cell lines does not involve UNG, but, instead, other DNA glycosylases that can excise uracil derivatives.

Dynamics of uracil and 5-fluorouracil in DNA.

The prodrug 5-fluorouracil (5-FU), after activation into 5-F-dUMP, is an extensively used anticancer agent that inhibits thymidylate synthase and leads to increases in dUTP and 5-F-dUTP levels in cells. One mechanism for 5-FU action involves DNA polymerase mediated incorporation of dUTP and 5-F-dUTP into genomic DNA leading to U/A, 5-FU/A, or 5-FU/G base pairs. These uracil-containing lesions are recognized and excised by several human uracil excision repair glycosylases (hUNG2, hSMUG2, and hTDG) leading to toxic abasic sites in DNA that may precipitate cell death.

Impact of linker strain and flexibility in the design of a fragment-based inhibitor.

The linking together of molecular fragments that bind to adjacent sites on an enzyme can lead to high-affinity inhibitors. Ideally, this strategy would use linkers that do not perturb the optimal binding geometries of the fragments and do not have excessive conformational flexibility that would increase the entropic penalty of binding. In reality, these aims are seldom realized owing to limitations in linker chemistry.

Uracil DNA glycosylase: revisiting substrate-assisted catalysis by DNA phosphate anions.

Uracil DNA glycosylase (UNG) is a powerful DNA repair enzyme that has been shown to stabilize a glycosyl cation reaction intermediate and a related tight binding inhibitor using electrostatic interactions with the +1 and -1, but not the +2, phosphodiester group of the single-stranded DNA substrate Ap (2+)Ap (1+)Up (1-)ApA. These experimental results differed considerably from computational findings using duplex DNA, where the +2 phosphate was found to stabilize the transition state by approximately 5 kcal/mol, suggesting that UNG uses different catalytic strategies with single-stranded and double-stranded DNA substrates. In addition, the computational studies indicated that the conserved and positively charged His148 (which hydrogen bonds to the +2 phosphate) destabilized the glycosyl cation intermediate by 6-8 kcal/mol through anticatalytic electrostatic interactions.

Enzymatic capture of an extrahelical thymine in the search for uracil in DNA.

The enzyme uracil DNA glycosylase (UNG) excises unwanted uracil bases in the genome using an extrahelical base recognition mechanism. Efficient removal of uracil is essential for prevention of C-to-T transition mutations arising from cytosine deamination, cytotoxic U*A pairs arising from incorporation of dUTP in DNA, and for increasing immunoglobulin gene diversity during the acquired immune response. A central event in all of these UNG-mediated processes is the singling out of rare U*A or U*G base pairs in a background of approximately 10(9) T*A or C*G base pairs in the human genome.

Mitigation of coagulation by removing clotting factors part 2: heparin-free extracorporeal circulation in a porcine model.

A subpopulation of patients would benefit from an anticoagulation strategy during extracorporeal circulation (ECC) that does not involve systemic administration of heparin and protamine. Inhibition of coagulation by adsorption of plasma clotting factors using protamine immobilized on a Sepharose matrix (PSM) has been explored. This investigation extends previous in vitro studies and demonstrates the feasibility of heparin-free ECC.

Mitigation of coagulation by removing clotting factors part 1: in vitro feasibility study.

Heparin is associated with adverse effects in some patients during extracorporeal circulation. A potential alternate anticoagulation strategy explored in this investigation involved mitigation of coagulation by removing clotting factors from blood by adsorption on a protamine-immobilized Sepharose matrix (PSM). Human or porcine plasmas treated with PSM in vitro were tested for clotting factors I (fibrinogen), II (prothrombin), VIII, and X, and proteins C and S, and for prothrombin time (PT), activated partial thromboplastin time (APTT), and total protein concentration.

Cohort study of 27 cases of endophthalmitis at a single institution.

Purpose: To identify potential risk factors associated with post-cataract surgery bacterial endophthalmitis.

Setting: The John A. Moran Eye Center, Salt Lake City, Utah, USA.

Implantation of an Artisan phakic intraocular lens for the correction of high myopia after penetrating keratoplasty.

We report 2 cases in which an Artisan phakic intraocular lens (IOL) (Ophtec) was used to successfully treat high myopia after penetrating keratoplasty (PKP). The first case was a 43-year-old man who had a manifest refraction of -13.75 +3.