https://www.cell.com/trends/biotechnology/pdf/S0167-7799(13)00087-5.pdf

ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering

RNA-guided engineered nucleases (RGENs) derived from the prokaryotic adaptive immune system known as CRISPR (clustered, regularly interspaced, short palindromic repeat)/Cas (CRISPR-associated) enable genome editing in human cell lines, animals, and plants, but are limited by off-target effects and unwanted integration of DNA segments derived from plasmids encoding Cas9 and guide RNA at both on-target and off-target sites in the genome. Here, we deliver purified recombinant Cas9 protein and guide RNA into cultured human cells including hard-to-transfect fibroblasts and pluripotent stem cells. RGEN ribonucleoproteins (RNPs) induce site-specific mutations at frequencies of up to 79%, while reducing off-target mutations associated with plasmid transfection at off-target sites that differ by one or two nucleotides from on-target sites. RGEN RNPs cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects. Furthermore, RNP delivery is less stressful to human embryonic stem cells, producing at least twofold more colonies than does plasmid transfection.

/pdf/highly-efficient-rna-guided-genome-editing-in-human-cells-1dwagla9ju.pdf

Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins

Summary: The Type II clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system is an adaptive immune response in prokaryotes, protecting host cells against invading phages or plasmids by cleaving these foreign DNA species in a targeted manner. CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs) enable genome editing in cultured cells, animals and plants, but are limited by off-target mutations. Here, we present a novel algorithm termed Cas-OFFinder that searches for potential off-target sites in a given genome or user-defined sequences. Unlike other algorithms currently available for identification of RGEN off-target sites, Cas-OFFinder is not limited by the number of mismatches and allows variations in protospacer-adjacent motif sequences recognized by Cas9, the essential protein component in RGENs. Cas-OFFinder is available as a command-line program or accessible via our website.

Availability and implementation: Cas-OFFinder free access at http://www.rgenome.net/cas-offinder.

Contact: rk.ca.uns@uaseab or rk.ca.uns@10miksj

/pdf/cas-offinder-a-fast-and-versatile-algorithm-that-searches-534naa4qkg.pdf

Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases

RNA-guided endonucleases (RGENs), derived from the prokaryotic adaptive immune system known as CRISPR/Cas, enable targeted genome engineering in cells and organisms. RGENs are ribonucleoproteins that consist of guide RNA and Cas9, a protein component originated from Streptococcus pyogenes. These enzymes cleave chromosomal DNA, whose sequence is complementary, to guide RNA in a targeted manner, producing site-specific DNA double-strand breaks (DSBs), the repair of which gives rise to targeted genome modifications. Despite broad interest in RGEN-mediated genome editing, these nucleases are limited by off-target mutations and unwanted chromosomal translocations associated with off-target DNA cleavages. Here, we show that off-target effects of RGENs can be reduced below the detection limits of deep sequencing by choosing unique target sequences in the genome and modifying both guide RNA and Cas9. We found that both the composition and structure of guide RNA can affect RGEN activities in cells to reduce off-target effects. RGENs efficiently discriminated on-target sites from off-target sites that differ by two bases. Furthermore, exome sequencing analysis showed that no off-target mutations were induced by two RGENs in four clonal populations of mutant cells. In addition, paired Cas9 nickases, composed of D10A Cas9 and guide RNA, which generate two single-strand breaks (SSBs) or nicks on different DNA strands, were highly specific in human cells, avoiding off-target mutations without sacrificing genome-editing efficiency. Interestingly, paired nickases induced chromosomal deletions in a targeted manner without causing unwanted translocations. Our results highlight the importance of choosing unique target sequences and optimizing guide RNA and Cas9 to avoid or reduce RGEN-induced off-target mutations.

/pdf/analysis-of-off-target-effects-of-crispr-cas-derived-rna-4qj3ty11fb.pdf

Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases

Programmable nucleases — including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided engineered nucleases (RGENs) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) system — enable targeted genetic modifications in cultured cells, as well as in whole animals and plants. The value of these enzymes in research, medicine and biotechnology arises from their ability to induce site-specific DNA cleavage in the genome, the repair (through endogenous mechanisms) of which allows high-precision genome editing. However, these nucleases differ in several respects, including their composition, targetable sites, specificities and mutation signatures, among other characteristics. Knowledge of nuclease-specific features, as well as of their pros and cons, is essential for researchers to choose the most appropriate tool for a range of applications.

A guide to genome engineering with programmable nucleases

A collection of TALENs targeted to 18,740 human protein-coding genes will facilitate genetic engineering of human cells.

A library of TAL effector nucleases spanning the human genome

Periodontitis is an inflammatory process that ultimately results in tooth loss. Although the primary etiologic agent for periodontitis is bacteria, the majority of periodontal tissue destruction is thought to be caused by an inappropriate host response. Reactive oxygen species (ROS) have been known to be involved in periodontal tissue destruction. We treated human gingival fibroblasts with lipopolysaccharide (LPS) obtained from E. coli and the periodontopathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis, and examined their inflammatory responses in the presence and absence of the antioxidant N-acetylcysteine (NAC). LPS enhanced ROS production, as well as, expression of pro-inflammatory cytokines such as interleukin-1beta, interleukin-6, interleukin-8 and tumor necrosis factor-alpha, and the production and activation of MMP2. NAC suppressed all LPS-induced inflammatory responses examined, suggesting that LPS-induced ROS may play a major regulatory role in these responses in gingival fibroblasts. In addition, NAC prevented LPS-induced activation of p38 MAPK and JNK but not phosphorylation and subsequent degradation of IkB. These results indicate that NAC exerts anti-inflammatory effects in LPS-stimulated gingival fibroblasts, functioning at least in part via down-regulation of JNK and p38 MAPK activation. Furthermore, this work suggests that antioxidants may be useful in adjunctive therapies that complement conventional periodontal treatments.

N-acetylcysteine prevents LPS-induced pro-inflammatory cytokines and MMP2 production in gingival fibroblasts

Objectives: To analyze the outcomes of a single- and a double-layered collagen membrane on the efficacy of onlay block grafts in terms of bone resorption and augmentation.

Material and methods: A total of 36 New Zealand white rabbits were used in this study. Calvarial bone blocks were obtained from one side of the parietal bone and fixed on the contaralateral side. The onlay grafts were covered with either no (C group), one (M1 group), or two (M2 group) layers of a non-cross-linked collagen membrane (BioGide®). After 2, 4, and 6 months of healing, rabbits were sacrificed and explanted specimens were prepared for histologic and histomorphometric analysis. At each period, the overall pattern of graft bone resorption and membrane biodegradation were examined histologically, and the sustained external form of grafted bone (%) and the remaining mineralized bone volume (%) were measured histomorphometrically.

Results: The M1 and M2 groups exhibited decreased bone resorption compared with the C group at all periods. The M2 group had less graft resorption and a higher bone density of the grafted bone than the M1 group. In the M1 group, the collagen membrane was degraded partially or completely by 4 months and was absent at 6 months. Conversely, for the M2 group, the membrane body was retained up to 6 months.

Conclusions: Our results demonstrated that the collagen membrane used here can reduce graft bone resorption. Furthermore, the double-layer technique using non-cross-linked collagen membranes (BioGide®) can enhance the efficacy of the onlay block bone graft technique in terms of both bone resorption and augmentation compared with a single-layer collagen membrane.

The efficacy of a double-layer collagen membrane technique for overlaying block grafts in a rabbit calvarium model

A library of TAL effector nucleases spanning the human genome. Nat Biotechnol

A new efficient synthetic method for chiral α-azido-α-alkylmalonates and α-aryloxy-α-alkylmalonates was developed. The enantioselective α-alkylation of diphenylmethyl tert-butyl α-bromomalonate under phase-transfer catalytic conditions [(S,S)-3,4,5-trifluorophenyl-NAS bromide, 50% KOH, toluene, and −40 °C) provided the corresponding α-bromo-α-alkylmalonates in high chemical yields (≤98%) and high optical yields (≤99% ee). The resulting α-alkylated products were converted to α-azido-α-alkylmalonates (≤96%, ≤97% ee) and α-aryloxy-α-alkylmalonates (≤79%, ≤93% ee) by SN2 substitution with sodium azide and aryloxides, respectively.

Enantioselective Synthesis of Chiral α-Azido and α-Aryloxy Quaternary Stereogenic Centers via the Phase-Transfer-Catalyzed α-Alkylation of α-Bromomalonates, Followed by SN2 Substitution

Doyoung Kim

Papers

A library of TAL effector nucleases spanning the human genome

N-acetylcysteine prevents LPS-induced pro-inflammatory cytokines and MMP2 production in gingival fibroblasts

The efficacy of a double-layer collagen membrane technique for overlaying block grafts in a rabbit calvarium model

A library of TAL effector nucleases spanning the human genome. Nat Biotechnol

Enantioselective Synthesis of Chiral α-Azido and α-Aryloxy Quaternary Stereogenic Centers via the Phase-Transfer-Catalyzed α-Alkylation of α-Bromomalonates, Followed by SN2 Substitution