gBlocks® Gene Fragments—Selected Citations

Hot Topic—CRISPR/Cas9 Complete Protocol

Yang L, Yang JL, et al
Curr Protoc Mol Biol, 107(31):1—17 (2014)

The discovery of the CRISPR/Cas9 system is a major advancement in targeted genome editing, heralding great potential for functional research and therapeutic applications. The system uses a short guide RNA (gRNA) to direct Cas9 to genomic sequences. Cas9 cleaves the target DNA producing either a deletion, or allowing for insertion of a new donor sequence by homologous recombination.

This publication from the lab of Dr George Church at Harvard University describes a complete protocol for designing gRNAs from gBlocks® Gene Fragments, and transferring the gRNA and Cas9 constructs into human-induced pluripotent stem cells and HEK293 cells. It also contains methods for assessing the success of the genome modification and isolating cells with the targeted changes. While the protocol is specific to 2 types of human cell lines, it should also provide useful information that is applicable to other cell culture models.

Recent Citation—Assembling Novel Transcripts

Marbiah MM, Harvey A, et al
EMBO J, [Epub ahead of print]: (2014)

In some cases, obtaining RNA or DNA for PCR cloning of a gene that is not well studied can be a significant challenge. In cases where the gene is critical to the study, sometimes the only option is to assemble the sequence using synthetically produced DNA.

In this paper, the Micalcl protein is one of several extracellular matrix proteins that are examined for their association with prion resistance. Cloned sequences for Micalcl were not readily available. As a fast and reliable alternative to finding or making template DNA, the authors assembled 5 gBlocks Gene Fragments to assemble the ~2 kb coding sequence for Micalcl, using the Gibson Assembly® method.

  1. Metabolic engineering of Saccharomyces cerevisiae to improve 1-hexadecanol production
    Xueyang Fenga, Jiazhang Liana, Huimin Zhao
    Metabolic Engineering, [In Press]:CRISPR/Cas9 Biofuels/Biomaterials (2014)
    gBlocks Gene Fragments were used to generate the fatty acyl-CoA reductase gene from barn owl.
    Biofuels/Biomaterials
  2. Highly efficient targeted chromosome deletions using CRISPR/Cas9
    He Z, Proudfoot C, et al
    Biotechnol Bioeng, [Epub ahead of print]: doi: 10.1002/bit.25490 (2014)
    gBlocks Gene Fragments are used to generate sgRNA sequences for targeting Cas9
    CRISPR/Cas9
  3. Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality
    Briner AE, Donohoue PD, et al
    Cell, [In press]:DOI: 10.1016/j.molcel.2014.09.019 (2014)
    gBlocks Gene Fragments were used to create sgRNA expression plasmids.
    CRISPR/Cas9
  4. Non-natural Olefin Cyclopropanation Catalyzed by Diverse Cytochrome P450s and Other Hemoproteins
    Heel T, McIntosh JA, et al
    Chembiochem, [Epub ahead of print]: (2014)
    gBlocks Gene Fragments were used to generate codon-optimized cytochrome P450 enzymes, and other hemoproteins, in order to study there catalytic use in non-natural olefin cyclopropanation reactions.
    Biofuels/Biomaterials
  5. The oil palm SHELL gene controls oil yield and encodes a homologue of SEEDSTICK
    Singh R, Low ET, et al
    Nature, 500(7462):340–344 (2013)
    gBlocks Gene Fragments are used to generate constructs for yeast two-hybrid assays.
    Biofuels/Biomaterials, Gibson Assembly Method
  6. Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals
    Runguphana W, Keasling J D
    Metabolic Engineering, online: (2013)

    3 codon-optimized gBlocks Gene Fragments were PCR assembled into a cloning vector to produce the wax ester synthase, atfA, for expression in S. cerevisiae.

    Biofuels/Biomaterials
  7. Programmable RNA recognition and cleavage by CRISPR/Cas9
    O'Connell MR, Oakes BL, et al
    Nature, [Epub ahead of print]: (2014)
    gBlocks Gene Fragments were used to create sgRNA, gene specific sequences.
    CRISPR/Cas9
  8. A Homology Integrated CRISPR-Cas (HI-CRISPR) system for one-step multi-gene disruptions in Saccharomyces cerevisiae
    Bao Z, Xiao H, et al
    ACS Synth Biol, [Epub ahead of print]: (2014)
    CRISPR/Cas9
  9. A G-quadruplex-containing RNA activates fluorescence in a GFP-like fluorophore
    Huang H, Suslov NB, et al
    Nat Chem Biol, [Epub ahead of print]: (2014)
    gBlocks Gene Fragmens were used to construct an expression construct for Spinach, an RNA aptamer that binds a GFP-like ligand and activates its green fluorescence.
  10. Shared VH1-46 gene usage by pemphigus vulgaris autoantibodies indicates common humoral immune responses among patients
    Cho MJ, Lo AS, et al
    Nat Commun, 5: (2014)
    gBlocks Gene Fragments were used to generate heavy and light antibody chains, and assembled using the Gibson Assembly® Method.
  11. Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila
    Port F, Chen HM, et al
    Proc Natl Acad Sci U S A, 111(29):2967–76 (2014)
    gBlocks Gene Fragments were used to construct a codon optimized Cas9 sequence for expression in Drosophila. Optimization of the sequence was performed using the IDT Codon Optimization tool.
    CRISPR/Cas9
  12. CRISPR/Cas9-Directed Genome Editing of Cultured Cells
    Yang L, Yang JL, et al
    Curr Protoc Mol Biol, 107(31):1—17 (2014)
    The authors provide a series of protocols for CRISPR/Cas9 genome editing in cell culture. The protocol describes how to use gBlocks Gene Fragments to generate the necessary guide RNAs and apply them to cell culture experiments.
    CRISPR/Cas9
  13. Cytotoxicity of the Vibrio vulnificus MARTX toxin Effector DUF5 is linked to the C2A Subdomain
    Antic I, Biancucci M, Satchell KJ
    Proteins, [Epub ahead of print]: (2014)
  14. An RNF168 fragment defective for focal accumulation at DNA damage is proficient for inhibition of homologous recombination in BRCA1 deficient cells
    Muñoz MC, Yanez DA, Stark JM
    Nucleic Acids Res, 42(12):7720–7733 (2014)
    gBlocks Gene Fragments were used to generate mutant variants of a Flag-tagged E3 ubiquitin ligase, RNF168.
  15. Comprehensive secondary structure elucidation of four genera of the family pospiviroidae
    Giguère T, Raj Adkar-Purushothama C, Perreault JP.
    PLoS One, 9(6): (2014)
  16. A “Push and Slide” Mechanism Allows Sequence-Insensitive Translocation of Secretory Proteins by the SecA ATPase
    Bauer BW, Shemesh T, et al
    Cell, 157(6):1416–1429 (2014)
  17. Assembly of highly standardized gene fragments for high-level production of porphyrins in E. coli
    Nielsen MT, Madsen KM, et al
    ACS Synth Biol, [Epub ahead of print]: (2014)
  18. Enhancing fatty acid ethyl ester production in Saccharomyces cerevisiae through metabolic engineering and medium optimization
    Thompson RA, Trinh CT
    Biotechnol Bioeng, [Epub ahead of print]: (2014)
  19. Hepatitis Delta Antigen Requires a Flexible Quasi-Double-Stranded RNA Structure to Bind and Condense Hepatitis Delta Virus RNA in a Ribonucleoprotein Complex.
    Griffin BL, Chasovskikh S, et al
    J Virol, [Epub ahead of print]: (2014)
    A gBlocks Gene Fragment was used to create a construct derived from sequences in the human herpesvirus 8 K12 gene, for use in transcribing the complementary RNA sequence, and with the sequence alterations allowing folding of the RNA in a manner similar to hepatitis delta virus RNA.
  20. GC-Rich DNA Elements Enable Replication Origin Activity in the Methylotrophic Yeast Pichia pastoris
    Liachko I, Youngblood RA. et al
    PLoS Genet, 10(3):e1004169 (2014)
    gBlocks Gene Fragments were used to generate mutant autonomously replicating sequences, or ARSs, associated with replication origins in yeast chromosomal DNA. 
    Mutagenesis
  21. Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system
    Ghorbal M, Gorman M, et al
    Nat Biotechnol, [Epub ahead of print]: (2014)
    A gBlocks Gene Fragment was used to create an sgRNA expression vector for use with the CRISPR/Cas9 system.
    CRISPR/Cas9
  22. Prosurvival Bcl-2 family members affect autophagy only indirectly, by inhibiting Bax and Bak
    Lindqvist LM, Heinlein M, et al
    PNAS, [Epub ahead of print]: (2014)
    Mutagenesis
  23. A diverse array of cancer-associated mTOR mutations are hyperactivating and can predict rapamycin sensitivity
    Grabiner BC, Nardi V, et al
    Cancer Discov, 4(5):554–563 (2014)
    Mutagenesis
  24. Hormone-Responsive Enhancer-Activity Maps Reveal Predictive Motifs, Indirect Repression, and Targeting of Closed Chromatin
    Shlyueva D, Stelzer C, et al
    Molecular Cell, 54(1):180–192 (2014)
    gBlocks Gene Fragments were used to synthesize mutant variants of the putative enhancer elements analyzed in this paper.
    Mutagenesis
  25. Design of synthetic yeast promoters via tuning of nucleosome architecture
    Curran KA, Crook NC, et al
    Nat Commun, 5: (2014)
  26. Identification of a gene regulatory network associated with prion replication
    Marbiah MM, Harvey A, et al
    EMBO J, [Epub ahead of print]: (2014)
    5 gBlocks Gene Fragments were used to assemble the ~2 kb coding sequence for Micalcl (NM_027587).
    Gibson Assembly Method
  27. Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly
    Torella JP, Boehm CR, et al
    Nucleic Acids Res, 42(1):681–689 (2014)
    gBlocks Gene Fragments were used to create basic part and destination vectors containing appropriate biologically inactive unique nucleotide sequences (UNSes). The 40 bp UNSes are designed to create the homologous overlaps that are compatible with the Gibson Assembly® Method, and make it possible to assemble multiple parts with repeated terminator and insulator sequences into functional synthetic circuits.
  28. A condition-specific codon optimization approach for improved heterologous gene expression in Saccharomyces cerevisiae
    Lanza AM, Curran KA, et al
    BMC Syst Biol, 8(33): (2014)
  29. Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool
    Ronda C, Pedersen LE, et al
    Biotechnol Bioeng, [Epub ahead of print]: (2014)
    A gBlocks Gene Fragment was used to generate an sgRNA expression cassette for use in CRISPR/Cas9 genome editing experiments in CHO cells.
    CRISPR/Cas9
  30. A conserved and essential basic region mediates tRNA binding to the Elp1 subunit of the Saccharomyces cerevisiae Elongator complex
    Di Santo R, Bandau S, Stark MJ.
    Mol Microbiol, [Epub ahead of print]: (2014)
    A gBlocks Gene Fragment was used to generate a nuclear localized, C-terminal GFP tag.
  31. Isolation of PCR quality microbial community DNA from heavily contaminated environments
    Gunawardana M, Chang S, et al
    J Microbiol Methods, [Epub ahead of print]: (2014)
    A gBlocks Gene Fragment was used in a serial dilution for the generation of a standard curve to convert Ct values into copy number for 16S rRNA.
    qPCR
  32. Dissection of thousands of cell type-specific enhancers identifies dinucleotide repeat motifs as general enhancer features
    Yanez-Cuna JO, Arnold CD, et al
    Genome Res, [Epub ahead of print]: (2014)
    gBlocks Gene Fragments were used to construct mutant versions of predicted enhancer elements to serve as negative controls in a luciferase reporter assay.
  33. Direct recognition of homology between double helices of DNA in Neurospora crassa
    Gladyshev E, Kleckner N
    Nat Commun, 5: (2014)
  34. Generation of human endometrial knockout cell lines with the CRISPR-Cas9 system confirms the prostaglandin F2α synthase activity of aldo-ketoreductase 1B1
    Lacroix Pépin N, Chapdelaine P, et al
    Mol Hum Reprod, [Epub ahead of print]: (2014)
    A gBlocks Gene Fragment was used to generate a single-guide RNA scaffold and cloned into a vector for use in CRISPR/Cas9 genome editing.
    CRISPR/Cas9
  35. Using synthetic templates to design an unbiased multiplex PCR assay
    Carlson CS, Emerson RO, et al
    Nat Commun, 4(2680):doi: 10.1038/ncomms3680 (2013)
    The authors have developed a multiplex PCR method that allows for quantitative analysis of T- and B- cell receptor diversity using next generation sequencing. gBlocks Gene Fragments are used as templates for optimization of the multiplex primer mix, a critical step for maintaining quantifiable differences and detecting low level transcripts.
    Next Generation Sequencing
  36. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing
    Esvelt KM, Mali P, et al
    Nat Methods, Epub ahead of print: (2013)
    The authors of this study, use IDT gBlocks® Gene Fragments to generate human optimized Cas9 orthologs from  S. thermophilus, N. meningitidis, and T. denticola, as well as tracRNA expression cassettes for each ortholog. The authors clarify the PAM requirements of the various enzymes, and show that combining Cas9 orthologs with a different PAM requirements can be used to carry out different tasks in parallel in bacterial and human cell types.
    CRISPR/Cas9
  37. Structure and Identification of a Pterin Dehydratase-like Protein as a Ribulose-bisphosphate Carboxylase/Oxygenase (RuBisCO) Assembly Factor in the α-Carboxysome
    Wheatley NM, Sundberg CD, et al
    J Biol Chem, 289:7973–7981 (2014)
    gBlocks Gene Fragments were used to generate codon-optimized acRAF genes from H. neapolitanus and P. marinus.
    Gibson Assembly Method
  38. The Mitochondrial Translocator Protein, TSPO, Inhibits HIV-1 Envelope Glycoprotein Biosynthesis via the Endoplasmic Reticulum-Associated Protein Degradation Pathway
    Zhou T, Dang Y, Zheng YH
    J Virol, 88(6):3474–3484 (2014)
    A gBlocks Gene Fragment was used to generate a single-guide RNA for use with the CRISPR/Cas9 system. The 455 bp fragment contained a U6 promoter, target sequence, and guide RNA scaffold.
    CRISPR/Cas9
  39. Antibody conjugation via one and two C-terminal selenocysteines
    Li X, Yang J, Rader C
    Methods, 65(1):133–138 (2014)
  40. Identification of Binding Sites in Huntingtin for the Huntingtin Interacting Proteins HIP14 and HIP14L
    Sanders SS, Mui KKN, et al
    PLOS ONE, [Epub ahead of print]: (2014)
  41. DNA Replicons for Plant Genome Engineering
    Baltes NJ, Gil-Humanes J, et al
    Plant Cell, [Epub ahead of print]: (2014)
  42. Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System
    Chen B, Gilbert LA, et al
    Cell, 155:1479–1491 (2013)
    gBlocks Gene Fragments were used to generate expression constructs for optimized sgRNAs, that were then used to target a nuclease-deficient, Cas9-GFP chimeric protein in chromatin imaging experiments.
    CRISPR/Cas9
  43. Validation of interleukin 28B genotyping assay for clinical use
    Cervinski MA, Sam SS, et al
    Clin Biochem, [Epub ahead of print]: (2014)
    gBlocks Gene Fragments were used as positive genotype controls for the described 5’ nuclease, SNP genotyping assay.
    SNP Genotyping
  44. An Optimized microRNA Backbone for Effective Single-Copy RNAi
    Fellmann C, Hoffmann T, et al
    Cell Rep, [Epub ahead of print]: (2013)
    gBlocks Gene Fragments were used to generate constructs with shRNA binding sites for a reporter assay to quantify shRNA knockdown.
  45. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH
    Brinkmann-Chen S, Flock T, et al
    Proc Natl Acad Sci U S A, 110(2):10946-10951 (2013)
  46. Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis
    Enyeart PJ, Mohr G, et al
    Mob DNA, 5(1): (2014)
  47. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library
    Koike-Yusa H, Li Y, et. al
    Nature biotechnol, [Epub ahead of print]: (2013)
  48. Computational design of a pH-sensitive IgG binding protein
    Strauch EM, Fleishman SJ, Baker D
    Proc Natl Acad Sci, [Epub ahead of print]: (2013)
  49. Creating functional engineered variants of the single-module non-ribosomal peptide synthetase IndC by T domain exchange
    Beer R, Herbst K, et al
    Mol. BioSyst., [Epub ahead of print]: (2014)
    gBlocks Gene Fragments were used to produce synthetic T domains that carry amino acids, which are then incorporated into peptide chains by non-ribosomal peptide synthetases.
  50. Repurposing CRISPR/Cas9 for in situ functional assays
    Malina A, Mills JR, et al
    Genes & Dev, 27:2602–2614 (2013)
    gBlocks Gene Fragments were used to generate a sgRNA constructs for in situ functional assays.
    CRISPR/Cas9
  51. A Human Torque Teno Virus Encodes a MicroRNA That Inhibits Interferon Signaling
    Kincaid RP, Burke JM, et al
    PLoS Pathog, doi: 10.1371/journal.ppat: (2013)
    gBlocks Gene Fragments were used to generate mutant and wild-type 3’UTR sequences that were used in a luciferase reporter assay to assess microRNA control of gene expression.
  52. Cell-free protein synthesis from a release factor 1 deficient Escherichia coli activates efficient and multiple site-specific non-standard amino acid incorporation
    Hong SH, Ntai I, et al
    ACS Synth Biol, [Epub ahead of print]: (2013)
  53. Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and λ repressors
    Priest DG, Cui L, et al
    Proc Natl Acad Sci U.S.A., [Epub ahead of print]: (2013)
  54. Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9
    Bassett AR, Tibbit C, et al
    Biol Open, [Epub ahead of print]: (2013)
    A gBlocks Gene Fragment was used to generate a sgRNA backbone, including a Drosophila U6 promoter.
    CRISPR/Cas9
  55. Retinoic acid-dependent regulation of miR-19 expression elicits vertebrate axis defects
    Franzosa JA, Bugel SM,
    FASEB J., [Epub ahead of print]: (2013)
    gBlocks Gene Fragments were used to generate 3' untranslated regions of the zebrafish cyp26a1 mRNA with putative miR-19 binding sites, and a corresponding mutated miR-19 binding site control.
  56. A Neuronal GPCR is Critical for the Induction of the Heat Shock Response in the Nematode C. elegans
    Maman M, Carvalhal Marques F, et al
    J Neurosci, 33(14):6102-6111 (2013)
  57. Translational control of Nrf2 within the open reading frame
    Perez-Leal O, Barrero CA, Merali S
    Biochem Biophys Res Commun, 437(1):134–139 (2013)
    A gBlocks Gene Fragment was used to replace codons in the open reading frame of the transcription factor Nrf2 with synonymous codons. The resulting construct was used to disrupt a post transcriptional regulation mechanism of Nrf2, while preserving the function of the translated protein.
  58. Controlling Macromolecular Topology with Genetically Encoded SpyTag-SpyCatcher Chemistry
    Zhang WB, Sun F, et al
    J Am Chem Soc, 138(37):13988-97 (2013)
    A gBlocks gene fragment was used to create the SpyCatcher motif. SpyTag–SpyCatcher chemistry can be used to direct the conformation of proteins and allows them to mimic properties of other synthetic polymers, making it a potentially useful system for development of new biomaterials. The SpyTag motif was generated with annealed oligonucleotides from IDT.
  59. Identification and Characterization of Novel Helicobacter pylori apo-Fur Regulated Target Genes
    Carpenter BM, Gilbreath JJ, et al
    J Bacteriol, 195:5526–5539 (2013)
    gBlocks Gene Fragments are used to create promoter variants with scrambled binding sites the apo-Fur transcriptional regulator, found in H. pylori. The scrambled promoters are used in a series of DNA binding and DNase I, footprinting studies.
  60. X-Box Binding Protein 1 (XBP1s) Is a Critical Determinant of Pseudomonas aeruginosa Homoserine Lactone- Mediated Apoptosis
    Valentine CD, Anderson MO, et al
    PLoS Pathog, 9(8):e1003576 (2013)

    gBlocks Gene Fragments were used to create leucine-to-alanine mutants of XBP1s and XBPΔ2 in the leucine zipper motif.

  61. Genomically recoded organisms expand biological functions
    Lajoie MJ, Rovner AJ, et al
    Science, 342(6156):357–60 (2013)
    gBlocks Gene Fragments were used to create GFP variants with either 1, 2, or 3 UAG stop codons within the GFP coding sequence. These GFP mutants were used to test genomically reprogrammed E. coli, and the reassigned function of the UAG codon for incorporating nonstandard amino acids.
  62. miR-125b targets erythropoietin and its receptor and their expression correlates with metastatic potential and ERBB2/HER2 expression
    Ferracin M, Bassi C, et al
    Mol Cancer, 12(1): [Epub ahead of print] (2013)
    gBlocks Gene Fragments are used to generate mutated 3'UTR deletions of the mir-125b seed regions found in both the erythropoietin and erythropoietin receptor mRNA sequences. The resulting constructs were cloned, and expressed in MCF7 and HEK-293 cells.
  63. RNA-guided gene activation by CRISPR-Cas9-based transcription factors
    Perez-Pinera P, Kocak DD, et al
    Nat Methods, Epub ahead of print: (2013)
    gBlocks Gene Fragments were used to generate crRNA and tracRNA expression cassettes, as well as a single-guide RNA expression cassette that was designed for easy cloning of future RNA spacer elements.
    CRISPR/Cas9
  64. RNA-guided human genome engineering via Cas9
    Mali P, Yang L, et. al
    Science, 336(6121):823–826 (2013)

    Use of gBlocks® Gene Fragments to assemble a sequence-optimized Cas9 for expression in human cells, as well as a Cas9_D10A variant.

    gBlocks, CRISPR/Cas9
  65. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system
    Cheng AW, Wang H, et al
    Cel Res, Epub ahead of print: (2013)
    A gBlocks Gene Fragment was used to add 10 repeats of the minimal VP16 transactivation domain, derived from herpes simplex virus, to a nuclease deficient Cas9. This allows the resulting dCas9VP160 chimeric protein to serve as a transcriptional activator that can be easily target by CRISPR machinery in a variety of cell types.
    CRISPR/Cas9
  66. Direct detection of alternative open reading frames translation products in human significantly expands the proteome
    Vanderperre B, Lucier JF, et al
    PLoS One, 8(8):1–12 (2013)
    gBlocks Gene Fragments were used to generate several plasmids to verify translation initiation from predicted AUG, start codons and the alternate proteins from the expressed mRNAs. The authors used multiple cloning methods, including the Gibson Assembly™ Method to generate these constructs.
    Gibson Assembly Method
  67. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering
    Mali P, Aach J, et al
    Nat Biotech, advance online publication: (2013)

    gBlocks Gene Fragments were used for creating sgRNA expression constructs for targeting mutant Cas9 variants to test either transcription activation or genome modification.

    CRISPR/Cas9
  68. Locus-specific editing of histone modifications at endogenous enhancers
    Mendenhall EM, Williamson KE, et al
    Nat Biotechnol, Epub ahead of print: (2013)
    A gBlocks Gene Fragment was used to add a 3X FLAG sequence tag, and a 29-amino-acid, serine-glycine linker to the C-terminal end of a TAL effector repeat in a plasmid cloning vector.
  69. Decaffeination and measurement of caffeine content by addicted Escherichia coli with a refactored N-demethylation operon from Pseudomonas putida CBB5
    Quandt EM, Hammerling MJ, et al
    ACS Synth Bio, [Epub ahead of print]:A–G (2013)
    gBlocks® Gene Fragments were assembled using the Gibson Assembly™ Method to generate some of the plasmid constructs involved in refactoring a N-demethylation operon that provides a caffeine degradation pathway from Pseudomonas putida CBB5 to E Coli, an organism more amenable to genetic engineering and industrial applications.The resulting cell lines requred caffeine to survive and their growth yield could serve as a biosensor to precisely measure caffeine concentrations, for example, in sodas and energy drinks. This work was part of a 2012 undergraduate iGEM project sponsored by IDT.
    Gibson Assembly Method
  70. Enhanced Primer Selection and Synthetic Amplicon Templates Optimize High-Resolution Melting Analysis of Single-Nucleotide Polymorphisms in a Large Population
    Bruzzone CM, Tawadros PS, et al
    Genet Test Mol Biomarkers, 17(9):675–80 (2013)

    Sequence-verified gBlocks® Gene Fragments were to verify the identity of homozygous peaks in this high-throughput, melt-curve analysis for SNP detection protocol.

    SNP Genotyping
  71. Systemic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay
    Kheradpour P, Ernst J, et al
    Genome Res, 23(5):800–811 (2013)

    gBlocks® Gene Fragments, directly cloned into PCR vectors and transfected into HepG2 and K562 cells, were used in luciferase assays to validate a massively parallel reporter assay that measures the effect of enhancer motifs on transcription.

  72. Barcoding cells using cell-surface programmable DNA-binding domains
    Mali P, Aach J, et al
    Nature Methods, [Epub ahead of print]:1–4 (2013)

    gBlocks® Gene Fragments were used to generate several surface zinc-finger DNA-binding domains (sZFs), this research group created cell lines that expressed the distinct domains on their surface. By then exposing the cells to fluor-conjugated dsDNA in a multi-step labeling approach, the scientists could essentially barcode the different cell lines. (Fluor-conjugated DNA probes (IDT) contained 4 phosphorothioate bonds on both the 5′ and 3′ ends to enhance protection against nucleases prevalent in extracellular medium.) The live cells could not only be tracked, but also specifically isolated and enriched from within a heterogeneous cell mixture.

  73. Splicing of internal large exons is defined by novel cis-acting sequence elements
    Bolisetty MT, Beemon KL
    Nucleic Acids Res, 40(18):1–11 (2012)
    gBlocks® Gene Fragments used as an alternative to site-directed mutagenesis to introduce 18 mutations spread over the 1039 nt exon 7 of the gene JARID2 in order to verify that C-rich consensus sites with a central invariant CA dinucleotide are important for the in splicing of large exons, >1000 nt.
  74. Differential role of axin RGS domain function in Wnt signaling during anteroposterior patterning and maternal axis formation
    Schneider PN, Slusarski DC, Houston DW
    PLoS One, 7(9):e44096 (2012)
    gBlocks® Gene Fragments used to construct an expression vector containing a hemagglutinin (HA)-tagged, APC peptide for verification that wild-type and mutant Axin-RGS domains do interact with APC, a central component of the β-catenin destruction complex.
  75. Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination
    Dickinson DJ, Ward JD, et al
    Nat Methods, Epub ahead of print: (2013)
    A codon-optimized Cas9 was created by ordering a series of overlapping gBlocks Gene Fragments, and assembled using the Gibson Assembly™ Method, and inserted into the vector pCFJ601. In addition, a single gBlocks Gene Fragment was inserted downstream of the Cas9 sequence, containing a U6 promoter, and the necessary elements for sgRNA function.
    CRISPR/Cas9, Gibson Assembly Method
  76. Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease
    Gratz SJ, Cummings AM, et al
    Genetics, Epub ahead of print: (2013)
    A gBlocks Gene Fragment was used to generate a chimeric single-guide RNA expression cassette with a U6 promoter that was designed for easy cloning of RNA spacer elements.
    CRISPR/Cas9
  77. CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes
    Gilbert LA, Larson MH, et al
    Cell, 154(2):422–51 (2013)
    gBlocks Gene Fragments were used to create an Mxi1 transcription repressor domain and assembled with a dCas9 construct using the Gibson Assembly™ method into an expression vector. The resulting chimeric, transcriptional repressor was then shown to be targetable using the CRISPR/Cas9 mechanism.
    CRISPR/Cas9, Gibson Assembly Method
  78. CRISPR/Cas9-Targeted Mutagenesis in Caenorhabditis elegans
    Waaijers S, Portegijs V, et al
    Genetics, Epub ahead of print: (2013)
    gBlocks Gene Fragments containing either a T7 or U6 promoter and the necessary tracrRNA and crRNA elements were used to create expression vectors for single guide RNAs (sgRNA) for targeting Cas9 to desired targets in C. elegans.
    CRISPR/Cas9
  79. Heritable genome editing in C. elegans via a CRISPR-Cas9 system
    Friedland A, Tzur Y, et al
    Nat Methods, Epub ahead of print: (2013)
    gBlocks Gene Fragments were used to create an RNA Pol III expression vector that was then used to create single guide RNAs (sgRNAs) for Cas 9 target genes, unc-119, dpy-13, klp1, and Y61A9LA.1 in C. elegans.
    CRISPR/Cas9
  80. Genetics and regulatory impact of alternative polyadenylation in human B-lymphoblastoid cells
    Yoon OK, Hsu TY, et al
    PLoS Genet, 8(8):e1002882 (2012)

    gBlocks® Gene Fragments used to generate a DNA standard for absolute quantification of mRNA from cells transfected with 3' UTR reporters.

    qPCR
  81. Generalized bacterial genome editing using mobile group II introns and Cre-lox
    Enyeart PJ, Chirieleison SM, et al
    Mol Syst Biol, 9:1–16 (2013)
  82. Do-it-yourself biology: challenges and promises for an open science and technology movement
    Landrain T, Meyer M, et al
    Systems and Synthetic Biology, online:1–12 (2013)
    Review describing advancements in synthetic biology that highlights the simplicity and affordability of using gBlocks Gene Fragments for assembling large, artificial genes.
  83. Integrase-defective Lentiviral Vectors as a Delivery Platform for Targeted Modification of Adenosine Deaminase Locus
    Joglekar AV, Hollis RP, et al
    Mol Ther, epub ahead of print: (2013)
    Overlapping gBlocks Gene Fragments were assembled to generate a codon-optimized zinc-finger nuclease, with 3xFLAG-tag and nuclear localization signal. Assembly was performed using spliced overlap PCR.
  84. Transcription of a cis-acting, Noncoding, Small RNA Is Required for Pilin Antigenic Variation in Neisseria gonorrhoeae
    Cahoon LA, Seifert HS
    PLoS Pathog, 9(1):e1003074 (2013)
    Use of gBlocks® Gene Fragments to create mutations that alter the direction and/or orientation of key promoter elements in the PilE bacterial gene, in Neisseria gonorrhoeae cells.