Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System. 2018

Ryan Marshall, and Colin S Maxwell, and Scott P Collins, and Thomas Jacobsen, and Michelle L Luo, and Matthew B Begemann, and Benjamin N Gray, and Emma January, and Anna Singer, and Yonghua He, and Chase L Beisel, and Vincent Noireaux
School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455, USA.

CRISPR-Cas systems offer versatile technologies for genome engineering, yet their implementation has been outpaced by ongoing discoveries of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation (TXTL) systems to vastly improve the speed and scalability of CRISPR characterization and validation. TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression-all without protein purification or live cells. We used TXTL to measure the dynamics of DNA cleavage and gene repression for single- and multi-effector CRISPR nucleases, predict gene repression strength in E. coli, determine the specificities of 24 diverse anti-CRISPR proteins, and develop a fast and scalable screen for protospacer-adjacent motifs that was successfully applied to five uncharacterized Cpf1 nucleases. These examples underscore how TXTL can facilitate the characterization and application of CRISPR technologies across their many uses.

UI MeSH Term Description Entries
D002474 Cell-Free System A fractionated cell extract that maintains a biological function. A subcellular fraction isolated by ultracentrifugation or other separation techniques must first be isolated so that a process can be studied free from all of the complex side reactions that occur in a cell. The cell-free system is therefore widely used in cell biology. (From Alberts et al., Molecular Biology of the Cell, 2d ed, p166) Cellfree System,Cell Free System,Cell-Free Systems,Cellfree Systems,System, Cell-Free,System, Cellfree,Systems, Cell-Free,Systems, Cellfree
D004269 DNA, Bacterial Deoxyribonucleic acid that makes up the genetic material of bacteria. Bacterial DNA
D004720 Endonucleases Enzymes that catalyze the hydrolysis of the internal bonds and thereby the formation of polynucleotides or oligonucleotides from ribo- or deoxyribonucleotide chains. EC 3.1.-. Endonuclease
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005818 Genetic Engineering Directed modification of the gene complement of a living organism by such techniques as altering the DNA, substituting genetic material by means of a virus, transplanting whole nuclei, transplanting cell hybrids, etc. Genetic Intervention,Engineering, Genetic,Intervention, Genetic,Genetic Interventions,Interventions, Genetic
D000094704 RNA, Guide, CRISPR-Cas Systems A component of CRISPR-Cas SYSTEMS. Cas endodeoxyribonucleases assemble with a guide RNA complex, a hybrid of CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) molecules, to form an active complex that cleaves DNA. crRNA and tracrRNA can be synthetically fused into a single RNA molecule, namely single guide RNA. Synthetic sgRNA is used with CRISPR-Cas SYSTEMS for targeted GENE EDITING. CRISPR Guide RNA,CRISPR-Cas Systems sgRNA (Single Guide RNA),Guide RNA (CRISPR-Cas Systems),Guide RNA, CRISPR-Cas Systems,RNA, CRISPR Guide,RNA, Guide (CRISPR-Cas Systems),RNA, Single Guide,RNA, Single-Guide,Single Guide RNA,Single-Guide RNA,Transactivating crRNA,crRNA,crRNA, Transactivating,sgRNA (CRISPR-Cas Systems),sgRNA (Single-Guide RNA),tracrRNA,Guide RNA, CRISPR,Guide RNA, CRISPR Cas Systems,Guide RNA, Single
D012275 Oryza A genus of grass family (Poaceae) that include several rice species. Oryza sativa,Rice,Rices
D014158 Transcription, Genetic The biosynthesis of RNA carried out on a template of DNA. The biosynthesis of DNA from an RNA template is called REVERSE TRANSCRIPTION. Genetic Transcription
D014176 Protein Biosynthesis The biosynthesis of PEPTIDES and PROTEINS on RIBOSOMES, directed by MESSENGER RNA, via TRANSFER RNA that is charged with standard proteinogenic AMINO ACIDS. Genetic Translation,Peptide Biosynthesis, Ribosomal,Protein Translation,Translation, Genetic,Protein Biosynthesis, Ribosomal,Protein Synthesis, Ribosomal,Ribosomal Peptide Biosynthesis,mRNA Translation,Biosynthesis, Protein,Biosynthesis, Ribosomal Peptide,Biosynthesis, Ribosomal Protein,Genetic Translations,Ribosomal Protein Biosynthesis,Ribosomal Protein Synthesis,Synthesis, Ribosomal Protein,Translation, Protein,Translation, mRNA,mRNA Translations
D064112 Clustered Regularly Interspaced Short Palindromic Repeats Repetitive nucleic acid sequences that are principal components of the archaeal and bacterial CRISPR-CAS SYSTEMS, which function as adaptive antiviral defense systems. CRISPR Arrays,CRISPR Clusters,CRISPR Elements,CRISPR Loci,CRISPR Locus,CRISPR Sequences,CRISPR Spacer Sequences,CRISPR Spacers,CRISPR-Cas Loci,CRISPRs,Clustered Regularly Interspaced Short Palindromic Repeat,Array, CRISPR,Arrays, CRISPR,CRISPR,CRISPR Array,CRISPR Cas Loci,CRISPR Cluster,CRISPR Element,CRISPR Sequence,CRISPR Spacer,CRISPR Spacer Sequence,CRISPR-Cas Locus,Cluster, CRISPR,Clusters, CRISPR,Element, CRISPR,Elements, CRISPR,Loci, CRISPR,Loci, CRISPR-Cas,Locus, CRISPR,Locus, CRISPR-Cas,Sequence, CRISPR,Sequence, CRISPR Spacer,Sequences, CRISPR,Sequences, CRISPR Spacer,Spacer Sequence, CRISPR,Spacer Sequences, CRISPR,Spacer, CRISPR,Spacers, CRISPR

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