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CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases. A high-fidelity variant of Streptococcus pyogenes CRISPR-Cas9 is reported that lacks detectable off-target events as assessed by genome-wide break capture and targeted sequencing methods. High-precision gene editing The CRISPR-Cas9 nucleases now widely used in gene editing can be readily customized, but can also induce substantial genome-wide off-target mutations at sequences that resemble the on-target site. Keith Joung and colleagues report a high-fidelity variant of Cas9 from Streptococcus pyogenes that shows on-target activities comparable to the wild-type enzyme, but with off-target events that are undetectable by genome-wide break capture and targeted sequencing methods., Author(s): Benjamin P. Kleinstiver [sup.1] [sup.2] , Vikram Pattanayak [sup.1] [sup.2] , Michelle S. Prew [sup.1] , Shengdar Q. Tsai [sup.1] [sup.2] , Nhu T. Nguyen [sup.1] , Zongli Zheng [...]

Site-specific gene addition can allow stable transgene expression for gene therapy. When possible, this is preferred over the use of promiscuously integrating vectors, which are sometimes associated with clonal expansion [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2009.11.030 Byline: Chunru Lin (1), Liuqing Yang (1), Bogdan Tanasa (1), Kasey Hutt (2), Bong-gun Ju (1)(5), Kenny Ohgi (1), Jie Zhang (1), David W. Rose (3), Xiang-Dong Fu (4), Christopher K. Glass (4), Michael G. Rosenfeld (1)(3) Keywords: HUMDISEASE; DNA; SIGNALING Abstract: Chromosomal translocations are a hallmark of leukemia/lymphoma and also appear in solid tumors, but the underlying mechanism remains elusive. By establishing a cellular model that mimics the relative frequency of authentic translocation events without proliferation selection, we report mechanisms of nuclear receptor-dependent tumor translocations. Intronic binding of liganded androgen receptor (AR) first juxtaposes translocation loci by triggering intra- and interchromosomal interactions. AR then promotes site-specific DNA double-stranded breaks (DSBs) at translocation loci by recruiting two types of enzymatic activities induced by genotoxic stress and liganded AR, including activation-induced cytidine deaminase and the LINE-1 repeat-encoded ORF2 endonuclease. These enzymes synergistically generate site-selective DSBs at juxtaposed translocation loci that are ligated by nonhomologous end joining pathway for specific translocations. Our data suggest that the confluence of two parallel pathways initiated by liganded nuclear receptor and genotoxic stress underlies nonrandom tumor translocations, which may function in many types of tumors and pathological processes. Author Affiliation: (1) Howard Hughes Medical Institute, University of California, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0648, USA (2) Bioinformatics Graduate Program, University of California, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0648, USA (3) Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0648, USA (4) Department of Cellular and Molecular Medicine, University of California, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0648, USA (5) Department of Life Science, Sogang University, Seoul 121-742, Korea Article History: Received 21 December 2008; Revised 20 April 2009; Accepted 11 November 2009 Article Note: (miscellaneous) Published online: December 3, 2009

Homing endonucleases stimulate gene conversion by generating site-specific DNA double-strand breaks that are repaired by homologous recombination. These enzymes are potentially valuable tools for targeted gene correction and genome engineering. We have engineered a variant of the I-Anil homing endonuclease that nicks its cognate target site. This variant contains a mutation of a basic residue essential for proton transfer and solvent activation in one active site. The cleavage mechanism, DNA-binding affinity, and substrate specificity profile of the nickase are similar to the wildtype enzyme. I-Anil nickase stimulates targeted gene correction in human cells, in cis and in trans, at [approximately equal to]1/4 the efficiency of the wild-type enzyme. The development of sequence-specific nicking enzymes like the I-Anil nickase will facilitate comparative analyses of DNA repair and mutagenesis induced by single- or double-strand breaks. protein engineering I recombination I single strand breaks I gene therapy I gene repair

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2005.06.008 Byline: Fritz Eckstein Abstract: RNA interference (RNAi) - inhibition of gene expression by small, non-coding RNAs [small interfering RNAs (siRNAs) or microRNAs (miRNAs)] - has changed our view of regulation of expression dramatically. The application of siRNAs for both functional analysis of genes and medication raises several questions. These include the design of the double-stranded oligonucleotides, their preparation and introduction into cells or animals either as chemically synthesized entities or as transcripts from a suitable vector. Delivery of the oligonucleotides, choice of vector, chemical modification to stabilize against nucleases and avoidance of side effects (e.g. stimulation of interferons) are major challenges. Work to identify the multiple targets of miRNAs is still in its infancy, and a clear distinction between siRNAs and miRNAs is difficult in some instances. Moreover, transcriptional silencing by RNAi is poorly understood; it is evident that the siRNA machinery is involved but the details await clarification. Given the multitude of interactions of the small non-coding RNAs revealed so far, we should be prepared to encounter, as yet, undiscovered interactions and mechanisms. Author Affiliation: Max-Planck-Institut fur experimentelle Medizin, Hermann-Rein-Str. 3, 37075 Gottingen, Germany

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2005.04.024 Byline: Marcin Nowotny (1), Sergei A. Gaidamakov (2), Robert J. Crouch (2), Wei Yang (1) Abstract: RNase H belongs to a nucleotidyl-transferase superfamily, which includes transposase, retroviral integrase, Holliday junction resolvase, and RISC nuclease Argonaute. We report the crystal structures of RNase H complexed with an RNA/DNA hybrid and a mechanism for substrate recognition and two-metal-ion-dependent catalysis. RNase H specifically recognizes the A form RNA strand and the B form DNA strand. Structure comparisons lead us to predict the catalytic residues of Argonaute and conclude that two-metal-ion catalysis is a general feature of the superfamily. In nucleases, the two metal ions are asymmetrically coordinated and have distinct roles in activating the nucleophile and stabilizing the transition state. In transposases, they are symmetrically coordinated and exchange roles to alternately activate a water and a 3'-OH for successive strand cleavage and transfer by a ping-pong mechanism. Author Affiliation: (1) Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 (2) Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892 Article History: Received 4 March 2005; Revised 7 April 2005; Accepted 19 April 2005 Article Note: (miscellaneous) Published: June 30, 2005

Research shows that all denatured forms of staphylococcal nuclease share a common nativelike 'topology' as indicated by the dipolar couplings following denaturation of the enzyme. Polyacrylamide gel electrophoresis reveals only small couplings but the sodium dodecyl sulfate/nuclease complexes show sharp and disperse (sup)1H--(sup)15N correlation spectra.

Research has been conducted on the carcinogen N-acetyl-2-aminofluorene forms of DNA adducts. Nuclease and protease digestion analyses have been evaluated.

Research has been conducted on the eukaryotic nuclease CEL I. Results indicate that CEL I exemplifies a new family of neutral pH magnesium-stimulated nucleases.

APN2 is the Saccharomyces cerevisiae homolog of the major human abasic (AP) endonuclease HAP1. Its role in repair of AP sites has been explored. These sites arise in DNA through spontaneous base loss. Damaged bases are removed by enzymes. APN1 (ital) encodes the major AP-endonuclease of the yeast. Human HAP1 (REF1) (ital) encodes the major AP endonuclease which acts as a redox regulatory protein. It also acts in DNA repair. There is evidence that Apn1 and Apn2 are alternate pathways for AP site repair. AP sites seem to be very cytotoxic and mutagenic in eukaryotes. The mutagenic bypass of AP sites seem to be mediated by the REV3, REV7-encoded DNA polymerase zeta.

An analysis of the triplet-state properties of several tryptophan analogues inserted in three nucleases of Staphylococcus is presented. Phosphorescence and optically detected magnetic resonance measurements have been done on the analogues 7-azatryptophan (7AW), 5-hydroxytryptophan (5HW) and 4-, 5-, and 6-fluorotryptophan (4FW, 5FW, 6FW). These analogues have been incorporated at position 140 of the wild-type nuclease, positions 66 and 140 of the V66W mutant and on the delta-137-149 fragment of the mutant.

Tryptophan (Trp) analogues have been inserted into Staphylococcus to study the guanidine-HCl induced unfolding of three types of nucleases. The three nucleases are the wild-type protein (WT), the V66W mutant and the delta-137-149 fragment of the mutant (V66W'). The Trp analogues used are 5-hydroxytryptophan (5HW), 7-azatryptophan (7AW), 4-fluorotryptophan (4FW), 5FW and 6FW. Results show that 5HW affects the stability of V66W' but not the WT nuclease. The 7AW analogue affects all the nucleases when substituted at positions 66 or 140. The three other analogues have negligible effect on the stability of the nucleases.

Tryptophan (Trp) analogues have been added into three nucleases of Staphylococcus to evaluate their use as intrinsic probes and their effect on the structure of the proteins. The nucleases are the the wild-type protein, its V66W mutant and the delta-137-149 fragment of the mutant. The wild-type protein has a single Trp at position 140, the V66W mutant has another Trp at position 66 and the delta-137-149 fragment has a Trp at position 66 only. The Trp analogues used include 5HW, 7AW, 4-fluorotryptophan (4FW), 5FW and 6FW.

RPP2, an essential gene that encodes a 15.8-kDa protein subunit of nuclear RNase P, has been identified in the genome of Saccharomyces cerevisiae. Rpp2 was detected by sequence similarity with a human protein, Rpp20, which copurifies with human RNase P. Epitope-tagged Rpp2 can be found in association with both RNase P and RNase mitochondrial RNA processing in immunoprecipitates from crude extracts of cells. Depletion of Rpp2 protein in vivo causes accumulation of precursor tRNAs with unprocessed introns and 5[prime] and 3[prime] termini, and leads to defects in the processing of the 35S precursor rRNA. Rpp2-depleted cells are defective in processing of the 5.8S rRNA. Rpp2 immunoprecipitates cleave both yeast precursor tRNAs and precursor tRNAs accurately at the expected sites and contain the Rpp1 protein orthologue of the human scleroderma autoimmune antigen, Rpp30. These results demonstrate that Rpp2 is a protein subunit of nuclear RNase P that is functionally conserved in eukaryotes from yeast to humans.

Optical and homonuclear nuclear magnetic resonance spectroscopy were utilized to characterize the water soluble polyammonium-substituted anthraquinone derivative, 27AQS2. RNA binding affinity studies have shown that the anthraquinone derivative interacted with DNA specifically at adenine and thymine regions of the minor groove. Mutation of 27AQS2 through radiation exposure resulted in lesser efficient cleavage.

The functional groups that mediate the catalytic activity of the leadzyme ribozyme were analyzed by the chemical synthesis of RNA using modified nucleotides. Analysis of the structure/function properties of leadzyme functional groups indicated the role of functional groups such as the N2 of G7 and the 2'-OH of G9 in facilitating the cooperativity between Pb2+ and leadzyme activity. Furthermore, Pb2+ binding site was detected in the vicinity of positions 3 and 4 of the asymmetric loop of the leadzyme.

Four variants of staphylococcal nuclease with potential disulfide bridges are studied by means of nuclear magnetic resonance to determine the energetic effects of disulfide bond formation. Results indicate that varying levels of folded state strain are induced by formation of the disulfide bridges. The data also suggest that the native the energetic effects can modulate the extent of successful stabilization.

Studies on excision repair of cisplatin-DNA adducts in a mammalian cell free system, show that 1,3d(GpTpG)-intrastrand adduct is repaired most efficiently. Proteins containing the high mobility group (HMG) domain DNA-binding motif can inhibit excision repair of the intrastrand 1,2-d(GpG) and -d(ApG) cross-links. This indicates that the inhibitory activity of HMG-domain proteins can be crucial in sensitivity of certain tumors to the drug. The difference in the repair rates of cisplatin-DNA cross-link are due to structural differences among the platinated DNA substrates.

The treatment of micrococcal nuclease with polyribosomes efficiently generates the stem-loop binding protein (SLBP) attached to the 3' end of histone mRNA as a 45-kDa monomer. The Northwestern blot assay suggests that 90% of SLBP in the cell is in ployribosomes and rest is in the nucleus. The Northwestern blot technique is based on a capacity to renature DNA binding proteins after their expression in bacteriophage vectors. SLBP appears to be involved in histone mRNA transport from the nucleus to the cytoplasm.

Determination of the structure of synthesized peptide B of staphylococcal nuclease by circular dichroism and 2D NMR spectroscopy reveals that the structure of the compact peptide resembles its structure in the intact protein. When the nascent helical structure in peptide B was stabilized, the CD spectra, chemical shift indexes, temperature coefficients and NOE data show the presence of a helix structure, which is identical to the structure in the native protein.

The ternary complexes of staphyllococcal nuclease with cobalt(II) and the mononucleotide possess different conformations in their metal-binding sites. In the first cobalt complex the cobalt ion is displaced 1.94 degree Angstroms from the common calcium position and the active site is possessed by a salt bridge between Asp-21 and Lys-70. The cobalt ion in the second structure binds with 0.36 degree Angstrom from the calcium position.

Time-resolved resonance energy transfer technique helps investigate the conformational flexibility of Staphylococcal nuclease mutant K45C. The lysine 45 is situated at the point where the flexible loop is replaced by a cysteine. The mutant K45C is involved in ligand or receptor binding in macromolecules and native proteins. Conformational flexibility occurs in a time scale of nanoseconds.

A heteronuclear NMR study of a denatured staphylococcal nuclease 131 residue fragment reveals that helix two and helix one retain characteristic helical structures, but also exhibit fast conformational changes between helical and extended conformations. The 83-86 and 94-97 segments exhibit properties associated with a native-like reverse-turn structure. The C-terminal alpha helix and the 5-strand beta-barrel indicate a hierarchical mechanism of nuclear folding.

Two nuclease assays facilitate the analysis of membrane-related nuclease activities in different mycoplasmal species. Nuclease activities may be necessary for development and survival of the organisms in hosts and occur in all mycoplasmal species. However, in Mycoplasma capricolum, magnesium is essential for its activity and it is restricted by calcium. Molecular weights of the nuclease proteins are measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis system.

Genetic analysis of the interaction between the lambda Gam protein and the individual subunits of RecBCD enzyme to determine whether the Gam protein interacts with any of the individual subunits was undertaken. The plasmids were injected into the strain. The presence of the thermoinducible prophage in IRB53 resulted in the transient induction of all the transformants using a heat treatment method that does not affect their colony-forming propensity but facilitates their production of Gam proteins.

A nuclease activity that is specific for G4 tetrastranded DNA was identified and characterized. This enzyme, found in a fraction partially purified from a yeast extract, requires the presence of a G4 structure and cleaves the DNA in a neighboring single-stranded region. The existence of this nucleolytic activity specific for a G4-DNA region suggests that this DNA structure may play a role in vivo.

A synthetic double-stranded DNA contaning a pyrimidine dimer at a distinct site was used to investigate pyrimidine dimer-dependent endonuclease activities from human cells. The DNA is incised at a unique site by human cell extracts. A cell extract prepared from excision repair-deficient xeroderma pigmentosum complementation group A is unable to incise the DNA, and extracts from complementation groups C, D and G incise the DNA at a different site than do normal human cell extracts. The results suggest that the incision reaction is associated with excision repair in human cells.

The purification and characterization of a zinc endonuclease from the Euglena gracilis nucleus were described. It binds to DNA, is nucleolytic and enhances the transcriptional rate of zinc RNA polymerase. The protein can also be isolated from zinc-deficient organisms, but it contains copper and not zinc. The copper-protein does not bind to or cleave DNA nor does it enhance transcription. Thus, copper replacement of zinc alters the function of the zincenzyme.

The backbone 1H and 15N resonances of unligated staphylococcal nuclease H124L were assigned by three-dimensional (3D) 1H-15N nuclear Overhauser- multiple-quantum coherence (NOESY-HMQC) nuclear magnetic resonance spectroscopy. Secondary structural elements (alpha-helices, beta-sheets, reverse turns and loops) were determined by analysis of patterns of NOE connectivities present in the 3D spectrum.

Accurate 1H, 13C and 15N chemical shifts were assigned for staphylococcal nuclease H124L. The chemical shits of unligated nuclease H124L were then compared with those of the nuclease-thymidine 3',5'-biphosphate-calcium (H124L.pdTp.Ca2+) ternary complex. The results show that backbone 15N, 1H(super N), 1H(super alpha) and 13C(super alpha) resonancesin certain sections of the amino acid sequence show large shifts upon binding pdTp and Ca2+.

A staphylococcal nuclease with deleted carboxyl terminus is characterized by nuclear magnetic resonance, circular dichroism andsmall angle x-ray scattering movements. The truncated nuclease exhibited a partly unfolded tertiary state with its secondary structure lacking persistencybut compact in physiological conditions. The nuclease returned to its folded conformation in the presence of 3',5'-bisphospho-2'-deoxythymidine. This characteristic proved the retained ability of the nuclease for renaturation.