Co-targeting strategy for precise,scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

Programmable site-specific nucleases, such as the clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas (Chlamydomonas reinhardtii). However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the dsDNA breaks induced by the RNPs is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. Here, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker)—in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precise edits in the homolog of bacterial ftsY or the WD and TetratriCopeptide repeats protein 1 genes in ∼1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways, or structures.

A transgene-free strategy allows precise editing of genes lacking a selectable phenotype by electroporation of CRISPR/Cas9 ribonucleoproteins and single-stranded oligodeoxynucleotide templates.     

Synthesis and structure-activity relationships of andrographolide analogues as novel cytotoxic agents

Andrographolide 1, the cytotoxic agent of the plant Andrographis paniculata was subjected to semi-synthetic studies leading to the preparation of a number of potent and novel analogues. Of the analogues synthesized, while 8,17-epoxy andrographolide 6 retained the cytotoxic activity of 1, ester derivatives of 6 exhibited considerable improvement in activity. Lower activity was observed when the epoxy moiety in the triacetate 9, derived from 6 was modified. Synthesis and structure-activity relationships are discussed.     

Protein Folding: A Few Random Thoughts

Abstract

Both the aqueous and lipid-induced structure of Kassinin, a dodecapeptide of amphibian origin, has been studied by two-dimensional proton nuclear magnetic resonance (2D ¹H-NMR) spectroscopy and distance geometry calculations. Unambiguous NMR assignments of protons have been made with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints obtained from the NMR data have been utilized in a distance geometry algorithm to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that, while in water Kassinin prefers to be in an extended chain conformation, in the presence of perdeuterated dodecylphosphocholine (DPC) micelles, a membrane model system, helical conformation is induced in the central core and C-terminal region (K4-M12) of the peptide. N-terminus though less defined also displays some degree of order and a possible turn structure. The conformation adopted by Kassinin in the presence of DPC micelles is consistent with the structural motif typical of neurokinin-1 selective agonists and with that reported for Eledoisin in hydrophobic environment.     

Crystal structures of MAP kinase p38 complexed to the docking sites on its nuclear substrate MEF2A and activator MKK3b

The structures of the MAP kinase p38 in complex with docking site peptides containing a phi(A)-X-phi(B) motif, derived from substrate MEF2A and activating enzyme MKK3b, have been solved. The peptides bind to the same site in the C-terminal domain of the kinase, which is both outside the active site and distinct from the "CD" domain previously implicated in docking site interactions. Mutational analysis on the interaction of p38 with the docking sites supports the crystallographic models and has uncovered two novel residues on the docking groove that are critical for binding. The two peptides induce similar large conformational changes local to the peptide binding groove. The peptides also induce unexpected and different conformational changes in the active site, as well as structural disorder in the phosphorylation lip.     

Enzymatic separation of cis and trans isomers of alicyclic alcohols

It has been discovered that phosphatases [alkaline phosphatase, orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1, and acid phosphatase, orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2] display a remarkable geometric specificity in the hydrolysis of cis and trans isomers of monoorthophosphate esters of substituted alicy clicalcohols. While steric hindrances prevent potato acid phosphatase from hydrolysing cis-2-methylcyclohexyl and cis-2-methylcyclopentyl phosphates, the corresponding trans isomers are readily hydrolysed by the enzyme (non-enzymatic, acid-catalysed or base-catalysed hydrolyses of the cis and trans isomers occur at similar rates). Cis isomers of methylcyclohexyl phosphates, in which the methyl group is remote from the hydrolysed ester bond, 3- or 4-, have nearly the same reactivities to phosphatases as their trans counterparts. However, if the methyl group in position 4 is replaced by a bulky substituent, e.g. tert-butyl, phosphatases again hydrolyse only the trans and not the cis isomer. These phenomena afford a simple method for preparative separation of cis and trans isomers of alicyclic alcohols: a mixture of the isomers is first phosphorylated with POCl₃ and then hydrolysed by phosphatase. The trans alcohol formed is extracted with CCl₄, followed by alkaline hydrolysis of the remaining cis-tester and subsequent extraction of the cis alcohol produced.     

The role of glycine (residue 89) in the central helix of EF-hand protein troponin-C exposed following amino-terminal alpha-helix deletion.

Because an N-terminal alpha-helical (N-helix) arm and a KGK-triplet (residues 88KGK90) in the central helix of troponin-C (TnC) are missing in calmodulin, several recent studies have attempted to elucidate the structure-function correlations of these units. Presently, with a family of genetically manipulated derivatives especially developed for this study and tested on permeabilized isolated single skeletal muscle fiber segments, we explored the specificities of the amino acid residues within the N-helix and the KGK-triplet in TnC. Noticeably, the amino acid compositions vary between the N-helices of the cardiac and skeletal TnC isoforms. On the other hand, the KGK-triplet is located similarly in both TnC isoforms. We previously indicated that deletion of the N-helix (mutant delta Nt) diminishes the tension obtained on activation with maximal calcium, but the contractile function is revived by the superimposed deletion of the 88KGK90-triplet (mutant delta Nt delta KGK; see Gulati J, Babu A, Su H, Zhang YF, 1993, J Biol Chem 268:11685-11690). Using this functional test, we find that replacement of Gly-89 with a Leu or an Ala could also overcome the contractile defect associated with N-helix deletion. On the other hand, replacement of the skeletal TnC N-helix with cardiac type N-helix was unable to restore contractile function. The findings indicate a destabilizing influence of Gly-89 residue in skeletal TnC and suggest that the N-terminal arm in normal TnC serves to moderate this effect. Moreover, specificity of the N-helix between cardiac and skeletal TnCs raises the possibility that resultant structural disparities are also important for the functional distinctions of the TnC isoforms.     

Adaptive developmental plasticity in methylene tetrahydrofolate reductase (MTHFR) C677T polymorphism limits its frequency in South Indians

Methylene tetrahydrofolate reductase (MTHFR) C677T polymorphism shows considerable heterogeneity in its distribution in humans worldwide. The current study was conducted to investigate whether this polymorphism exhibited adaptive developmental plasticity in the control of the TT-genotype frequency. We screened 1,818 South Indian subjects (895 males and 923 females) for MTHFR C677T polymorphism using PCR–restriction fragment length polymorphism approach. MTHFR 677T-allele frequency in males and females was 9.1 and 11.0 %, respectively. Compared to females, males had lower frequency of TT-genotype [odds ratio 0.31, 95 % confidence interval (CI) 0.08–1.01]. The frequency of MTHFR 677T-allele was highest in the age group of 20–40 years and it gradually decreased from 40–60 to 60–80 years (P _trend <0.0001). MTHFR 677TT-genotype was associated with 7.02-folds (95 % CI: 2.12–25.63, P < 0.0001) cumulative risk for recurrent pregnancy loss (RPL), neural tube defects (NTDs) and deep vein thrombosis (DVT). Linear regression model suggested that male gender exhibited increased homocysteine levels by 9.35 μmol/L while each MTHFR 677T-allele contributed to 4.63 μmol/L increase in homocysteine. Plasma homocysteine showed inverse correlation with dietary folate (r = ?0.17, P < 0.0001), B₂ (r = ?0.14, P < 0.0001) and B₆ (r = ?0.07, P = 0.03). Examination of the spontaneously aborted fetuses (n = 35) showed no significant association of fetal genotype on its in utero viability. From the current study, it was concluded that C677T seemed to have acquired adaptive developmental plasticity among South Indians due to environmental influences thus contributing to hyperhomocysteinemia and its associated complications such as RPL, NTDs, DVT, etc.     

Novel reactivity aspects of cis-bis(1-alkenyl)platinum(II) compounds of the type Pt(CH2(CH2)nCHCH2)2L2 (where L2 = dppp, dppe, dppm and n = 1, 2)

We describe the synthesis, structure and reactivity of novel bis(1-alkenyl)platinum(II) complexes, Pt[CH₂(CH₂)_nCHCH₂]₂L₂ (where L₂ = dppp, dppe, dppm and n = 1, 2). The stability of the title complexes with the different ligands is discussed. The steric, chelating and electronic properties of the ligands have a significant impact on the structure as well as on the reactivity of the complexes. Novel reactions with elemental sulfur and carbon dioxide are described and discussed.