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Nature Communications Dec 2018The creation of genome-wide libraries for CRISPR knockout (CRISPRko), interference (CRISPRi), and activation (CRISPRa) has enabled the systematic interrogation of gene...
The creation of genome-wide libraries for CRISPR knockout (CRISPRko), interference (CRISPRi), and activation (CRISPRa) has enabled the systematic interrogation of gene function. Here, we show that our recently-described CRISPRko library (Brunello) is more effective than previously published libraries at distinguishing essential and non-essential genes, providing approximately the same perturbation-level performance improvement over GeCKO libraries as GeCKO provided over RNAi. Additionally, we present genome-wide libraries for CRISPRi (Dolcetto) and CRISPRa (Calabrese), and show in negative selection screens that Dolcetto, with fewer sgRNAs per gene, outperforms existing CRISPRi libraries and achieves comparable performance to CRISPRko in detecting essential genes. We also perform positive selection CRISPRa screens and demonstrate that Calabrese outperforms the SAM approach at identifying vemurafenib resistance genes. We further compare CRISPRa to genome-scale libraries of open reading frames (ORFs). Together, these libraries represent a suite of genome-wide tools to efficiently interrogate gene function with multiple modalities.
Topics: CRISPR-Associated Protein 9; CRISPR-Cas Systems; Genomic Library; Streptococcus pyogenes
PubMed: 30575746
DOI: 10.1038/s41467-018-07901-8 -
Cell Systems Mar 2017A systems-level understanding of Gram-positive bacteria is important from both an environmental and health perspective and is most easily obtained when high-quality,...
A systems-level understanding of Gram-positive bacteria is important from both an environmental and health perspective and is most easily obtained when high-quality, validated genomic resources are available. To this end, we constructed two ordered, barcoded, erythromycin-resistance- and kanamycin-resistance-marked single-gene deletion libraries of the Gram-positive model organism, Bacillus subtilis. The libraries comprise 3,968 and 3,970 genes, respectively, and overlap in all but four genes. Using these libraries, we update the set of essential genes known for this organism, provide a comprehensive compendium of B. subtilis auxotrophic genes, and identify genes required for utilizing specific carbon and nitrogen sources, as well as those required for growth at low temperature. We report the identification of enzymes catalyzing several missing steps in amino acid biosynthesis. Finally, we describe a suite of high-throughput phenotyping methodologies and apply them to provide a genome-wide analysis of competence and sporulation. Altogether, we provide versatile resources for studying gene function and pathway and network architecture in Gram-positive bacteria.
Topics: Amino Acids; Bacillus subtilis; Gene Deletion; Gene Library; Genomic Library; Genomics; High-Throughput Screening Assays; Sequence Deletion; Spores, Bacterial
PubMed: 28189581
DOI: 10.1016/j.cels.2016.12.013 -
Nature Methods Jun 2011Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss-of-function and gain-of-function experiments....
Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss-of-function and gain-of-function experiments. We describe the production of a sequence-confirmed, clonal collection of over 16,100 human open-reading frames (ORFs) encoded in a versatile Gateway vector system. Using this ORFeome resource, we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.
Topics: Cloning, Molecular; Genetic Vectors; Genomic Library; Humans; Lentivirus; Open Reading Frames
PubMed: 21706014
DOI: 10.1038/nmeth.1638 -
Nature Biotechnology Apr 2023Large serine recombinases (LSRs) are DNA integrases that facilitate the site-specific integration of mobile genetic elements into bacterial genomes. Only a few LSRs,...
Large serine recombinases (LSRs) are DNA integrases that facilitate the site-specific integration of mobile genetic elements into bacterial genomes. Only a few LSRs, such as Bxb1 and PhiC31, have been characterized to date, with limited efficiency as tools for DNA integration in human cells. In this study, we developed a computational approach to identify thousands of LSRs and their DNA attachment sites, expanding known LSR diversity by >100-fold and enabling the prediction of their insertion site specificities. We tested their recombination activity in human cells, classifying them as landing pad, genome-targeting or multi-targeting LSRs. Overall, we achieved up to seven-fold higher recombination than Bxb1 and genome integration efficiencies of 40-75% with cargo sizes over 7 kb. We also demonstrate virus-free, direct integration of plasmid or amplicon libraries for improved functional genomics applications. This systematic discovery of recombinases directly from microbial sequencing data provides a resource of over 60 LSRs experimentally characterized in human cells for large-payload genome insertion without exposed DNA double-stranded breaks.
Topics: Humans; Integrases; Genome, Human; Genetic Engineering; Transfection; Genomic Library
PubMed: 36217031
DOI: 10.1038/s41587-022-01494-w -
The Journal of Heredity May 2021We present a protocol to prepare extracted DNA for sequencing on the Illumina sequencing platform that has been optimized for ancient and degraded DNA. Our approach, the...
We present a protocol to prepare extracted DNA for sequencing on the Illumina sequencing platform that has been optimized for ancient and degraded DNA. Our approach, the Santa Cruz Reaction or SCR, uses directional splinted ligation of Illumina's P5 and P7 adapters to convert natively single-stranded DNA and heat denatured double-stranded DNA into sequencing libraries in a single enzymatic reaction. To demonstrate its efficacy in converting degraded DNA molecules, we prepare 5 ancient DNA extracts into sequencing libraries using the SCR and 2 of the most commonly used approaches for preparing degraded DNA for sequencing: BEST, which targets and converts double-stranded DNA, and ssDNA2.0, which targets and converts single-stranded DNA. We then compare the efficiency with which each approach recovers unique molecules, or library complexity, given a standard amount of DNA input. We find that the SCR consistently outperforms the BEST protocol in recovering unique molecules and, despite its relative simplicity to perform and low cost per library, has similar performance to ssDNA2.0 across a wide range of DNA inputs. The SCR is a cost- and time-efficient approach that minimizes the loss of unique molecules and makes accessible a taxonomically, geographically, and a temporally broader sample of preserved remains for genomic analysis.
Topics: DNA, Ancient; Gene Library; Genomic Library; High-Throughput Nucleotide Sequencing; Sequence Analysis, DNA
PubMed: 33768239
DOI: 10.1093/jhered/esab012 -
Methods (San Diego, Calif.) Oct 2010Genomic SELEX is a discovery tool for genomic aptamers, which are genomically encoded functional domains in nucleic acid molecules that recognize and bind specific... (Review)
Review
Genomic SELEX is a discovery tool for genomic aptamers, which are genomically encoded functional domains in nucleic acid molecules that recognize and bind specific ligands. When combined with genomic libraries and using RNA-binding proteins as baits, Genomic SELEX used with high-throughput sequencing enables the discovery of genomic RNA aptamers and the identification of RNA-protein interaction networks. Here we describe how to construct and analyze genomic libraries, how to choose baits for selections, how to perform the selection procedure and finally how to analyze the enriched sequences derived from deep sequencing. As a control procedure, we recommend performing a "Neutral" SELEX experiment in parallel to the selection, omitting the selection step. This control experiment provides a background signal for comparison with the positively selected pool. We also recommend deep sequencing the initial library in order to facilitate the final in silico analysis of enrichment with respect to the initial levels. Counter selection procedures, using modified or inactive baits, allow strengthening the binding specificity of the winning selected sequences.
Topics: Aptamers, Nucleotide; Chromosome Mapping; Genomic Library; RNA; SELEX Aptamer Technique
PubMed: 20541015
DOI: 10.1016/j.ymeth.2010.06.004 -
Proceedings of the National Academy of... Oct 1989Four Drosophila melanogaster homeobox genes were found by screening a genomic DNA library with oligodeoxynucleotides that correspond to a conserved amino acid sequence... (Comparative Study)
Comparative Study
Four Drosophila melanogaster homeobox genes were found by screening a genomic DNA library with oligodeoxynucleotides that correspond to a conserved amino acid sequence that is part of the putative of homeobox proteins that recognizes nucleotide sequences in DNA. The amino acid sequences of NK-2, NK-3, and NK-4 homeoboxes are more closely related to one another (59-66% homology) than they are to other Drosophila homeoboxes (28-54% homology), whereas the homeobox of NK-1 is most closely related, in order of decreasing homology, to muscle segment homeobox, zerknüllt-1, NK-3, and distal-less homeoboxes. Three of the genes, NK-1, NK-3, and NK-4, comprise a cluster of homeobox genes located in the 93E1-5 region of the right arm of the third chromosome, whereas the fourth homeobox gene, NK-2, is located in the 1C1-5 region of the X chromosome.
Topics: Amino Acid Sequence; Animals; Base Sequence; Chromosome Mapping; Cloning, Molecular; DNA-Binding Proteins; Drosophila melanogaster; Genes; Genes, Homeobox; Genomic Library; Molecular Sequence Data; Oligonucleotide Probes; Restriction Mapping; Sequence Homology, Nucleic Acid
PubMed: 2573058
DOI: 10.1073/pnas.86.20.7716 -
Developmental Dynamics : An Official... Apr 2005The recent sequencing and draft assembly of a chicken genome has provided biologists with an invaluable research tool that complements a growing list of additional avian... (Review)
Review
The recent sequencing and draft assembly of a chicken genome has provided biologists with an invaluable research tool that complements a growing list of additional avian genomic resources. For many researchers, finding and using these resources is challenging, because information is presented through an increasing number of Web sites and browser navigation frequently requires specific knowledge and expertise. This primer provides an overview of online genomic resources for the chicken, including the Ensembl, UCSC, and NCBI annotated chicken genome browsers; expressed sequence tag and in situ hybridization databases; and sources for microarrays, cDNAs, and bacterial artificial chromosomes (BACs). Several short tutorials oriented toward the biologist with limited bioinformatics skills outline how to retrieve several types of commonly needed information and reagents.
Topics: Animals; Chickens; Chromosomes, Artificial, Bacterial; Databases, Nucleic Acid; Expressed Sequence Tags; Genomic Library; Sequence Analysis, DNA
PubMed: 15739221
DOI: 10.1002/dvdy.20339 -
Genome Biology Jan 2021CRISPR guide RNA libraries have been iteratively improved to provide increasingly efficient reagents, although their large size is a barrier for many applications. We...
CRISPR guide RNA libraries have been iteratively improved to provide increasingly efficient reagents, although their large size is a barrier for many applications. We design an optimised minimal genome-wide human CRISPR-Cas9 library (MinLibCas9) by mining existing large-scale gene loss-of-function datasets, resulting in a greater than 42% reduction in size compared to other CRISPR-Cas9 libraries while preserving assay sensitivity and specificity. MinLibCas9 provides backward compatibility with existing datasets, increases the dynamic range of CRISPR-Cas9 screens and extends their application to complex models and assays.
Topics: CRISPR-Cas Systems; Gene Library; Genome, Human; Genome-Wide Association Study; Genomic Library; Humans; Organoids; RNA, Guide, CRISPR-Cas Systems
PubMed: 33478580
DOI: 10.1186/s13059-021-02268-4 -
Proceedings of the National Academy of... Aug 1999Complete genome sequences are providing a framework to allow the investigation of biological processes by the use of comprehensive approaches. Genome analysis also is...
Complete genome sequences are providing a framework to allow the investigation of biological processes by the use of comprehensive approaches. Genome analysis also is having a dramatic impact on medicine through its identification of genes and mutations involved in disease and the elucidation of entire microbial gene sets. Studies of the sequences of model organisms, such as that of the nematode worm Caenorhabditis elegans, are providing extraordinary insights into development and differentiation that aid the study of these processes in humans. The field of functional genomics seeks to devise and apply technologies that take advantage of the growing body of sequence information to analyze the full complement of genes and proteins encoded by an organism.
Topics: Animals; Caenorhabditis; DNA; Genetics; Genome, Human; Genomic Library; Humans; Proteins; RNA; Saccharomyces cerevisiae
PubMed: 10430853
DOI: 10.1073/pnas.96.16.8825