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1.
Methods Enzymol ; 629: 493-511, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31727255

RESUMO

CRISPR-Cas systems naturally rely on CRISPR arrays to achieve immunity against multiple foreign invaders, where these arrays are also being utilized for multiplexed targeting as part of CRISPR technologies. However, CRISPR arrays have proven difficult to synthesize or assemble to-date due to the repetitive DNA repeats in each array. To overcome this barrier, we recently reported a cloning method we term CRATES (CRISPR Assembly through Trimmed Ends of Spacers) for the single-step, efficient generation of large Class 2 CRISPR arrays. CRATES generates CRISPR arrays through assembly of multiple repeat-spacer subunits using defined junction sequences within the trimmed portion of the CRISPR spacers. These arrays can be utilized by single-effector nucleases associated with Class 2 CRISPR-Cas systems, such as Cas9, Cas12a/Cpf1, or Cas13a/C2c2. Here, we describe in detail the steps for generating arrays utilized by Cas9 and Cas12a as well as composite arrays co-utilized by both nucleases. We also generate a representative three-spacer array and demonstrate multiplexed DNA cleavage through plasmid-clearance assays in Escherichia coli. This method is expected to simplify the study of natural CRISPR arrays and facilitate multiplexed targeting with programmable nucleases from Class 2 Cas nucleases across the myriad applications of CRISPR technologies.

2.
Nat Commun ; 10(1): 2948, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270316

RESUMO

CRISPR-Cas systems inherently multiplex through CRISPR arrays-whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Biblioteca Gênica , Técnicas Genéticas , RNA/biossíntese , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , DNA/genética , Endonucleases/metabolismo , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Plasmídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo
3.
FEMS Microbiol Lett ; 366(8)2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31004485

RESUMO

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nuclease Acidaminococcus sp. Cas12a (AsCas12a, also known as AsCpf1) has become a popular alternative to Cas9 for genome editing and other applications. AsCas12a has been associated with a TTTV protospacer-adjacent motif (PAM) as part of target recognition. Using a cell-free transcription-translation (TXTL)-based PAM screen, we discovered that AsCas12a can also recognize GTTV and, to a lesser degree, GCTV motifs. Validation experiments involving DNA cleavage in TXTL, plasmid clearance in Escherichia coli, and indel formation in mammalian cells showed that AsCas12a was able to recognize these motifs, with the GTTV motif resulting in higher cleavage efficiency compared to the GCTV motif. We also observed that the -5 position influenced the activity of DNA cleavage in TXTL and in E. coli, with a C at this position resulting in the lowest activity. Together, these results show that wild-type AsCas12a can recognize non-canonical GTTV and GCTV motifs and exemplify why the range of PAMs recognized by Cas nucleases are poorly captured with a consensus sequence.

4.
RNA Biol ; : 1-9, 2018 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-30252595

RESUMO

The Class 2 Type V-A CRISPR effector protein Cas12a/Cpf1 has gained widespread attention in part because of the ease in achieving multiplexed genome editing, gene regulation, and DNA detection. Multiplexing derives from the ability of Cas12a alone to generate multiple guide RNAs from a transcribed CRISPR array encoding alternating conserved repeats and targeting spacers. While array design has focused on how to optimize guide-RNA sequences, little attention has been paid to sequences outside of the CRISPR array. Here, we show that a structured hairpin located immediately downstream of the 3' repeat interferes with utilization of the adjacent encoded guide RNA by Francisella novicida (Fn)Cas12a. We first observed that a synthetic Rho-independent terminator immediately downstream of an array impaired DNA cleavage based on plasmid clearance in E. coli and DNA cleavage in a cell-free transcription-translation (TXTL) system. TXTL-based cleavage assays further revealed that inhibition was associated with incomplete processing of the transcribed CRISPR array and could be attributed to the stable hairpin formed by the terminator. We also found that the inhibitory effect partially extended to upstream spacers in a multi-spacer array. Finally, we found that removing the terminal repeat from the array increased the inhibitory effect, while replacing this repeat with an unprocessable terminal repeat from a native FnCas12a array restored cleavage activity directed by the adjacent encoded guide RNA. Our study thus revealed that sequences surrounding a CRISPR array can interfere with the function of a CRISPR nuclease, with implications for the design and evolution of CRISPR arrays.

5.
PLoS One ; 12(9): e0184664, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28910343

RESUMO

E. coli bacteria move in streams freely in a planktonic state or attached to suspended particulates. Attachment is a dynamic process, and the fraction of attached microorganisms is thought to be affected by both bacterial characteristics and particulate properties. In this study, we investigated how the properties of cell surfaces and stream particulates influence attachment. Attachment assays were conducted for 77 E. coli strains and three model particulates (ferrihydrite, Ca-montmorillonite, or corn stover) under environmentally relevant conditions. Surface area, particle size distribution, and total carbon content were determined for each type of particulate. Among the three particulates, attachment fractions to corn stover were significantly larger than the attachments to 2-line ferrihydrite (p-value = 0.0036) and Ca-montmorillonite (p-value = 0.022). Furthermore, attachment to Ca-montmorillonite and corn stover was successfully modeled by a Generalized Additive Model (GAM) using cell characteristics as predictor variables. The natural logarithm of the net charge on the bacterial surface had a significant, positive, and linear impact on the attachment of E. coli bacteria to Ca-montmorillonite (p-value = 0.013), but it did not significantly impact the attachment to corn stover (p-value = 0.36). The large diversities in cell characteristics among 77 E. coli strains, particulate properties, and attachment fractions clearly demonstrated the inadequacy of using a static parameter or linear coefficient to predict the attachment behavior of E. coli in stream water quality models.


Assuntos
Aderência Bacteriana , Escherichia coli/fisiologia , Algoritmos , Sítios de Ligação Microbiológicos , Bentonita , Tamanho da Partícula , Propriedades de Superfície
6.
Front Microbiol ; 8: 708, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28515712

RESUMO

Understanding the genetic factors that govern microbe-sediment interactions in aquatic environments is important for water quality management and reduction of waterborne disease outbreaks. Although chemical properties of bacteria have been identified that contribute to initiation of attachment, the outer membrane proteins that contribute to these chemical properties still remain unclear. In this study we explored the attachment of 78 Escherichia coli environmental isolates to corn stover, a representative agricultural residue. Outer membrane proteome analysis led to the observation of amino acid variations, some of which had not been previously described, in outer membrane protein A (OmpA) at 10 distinct locations, including each of the four extracellular loops, three of the eight transmembrane segments, the proline-rich linker and the dimerization domain. Some of the polymorphisms within loops 1, 2, and 3 were found to significantly co-occur. Grouping of sequences according to the outer loop polymorphisms revealed five distinct patterns that each occur in at least 5% of our isolates. The two most common patterns, I and II, are encoded by 33.3 and 20.5% of these isolates and differ at each of the four loops. Statistically significant differences in attachment to corn stover were observed among isolates expressing different versions of OmpA and when different versions of OmpA were expressed in the same genetic background. Most notable was the increased corn stover attachment associated with a loop 3 sequence of SNFDGKN relative to the standard SNVYGKN sequence. These results provide further insight into the allelic variation of OmpA and implicate OmpA in contributing to attachment to corn stover.

7.
Front Microbiol ; 7: 1732, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27847507

RESUMO

The importance of E. coli as an indicator organism in fresh water has led to numerous studies focusing on cell properties and transport behavior. However, previous studies have been unable to assess if differences in E. coli cell surface properties and genomic variation are associated with different environmental habitats. In this study, we investigated the variation in characteristics of E. coli obtained from stream water and stream bottom sediments. Cell properties were measured for 77 genomically different E. coli strains (44 strains isolated from sediments and 33 strains isolated from water) under common stream conditions in the Upper Midwestern United States: pH 8.0, ionic strength 10 mM and 22°C. Measured cell properties include hydrophobicity, zeta potential, net charge, total acidity, and extracellular polymeric substance (EPS) composition. Our results indicate that stream sediment E. coli had significantly greater hydrophobicity, greater EPS protein content and EPS sugar content, less negative net charge, and higher point of zero charge than stream water E. coli. A significant positive correlation was observed between hydrophobicity and EPS protein for stream sediment E. coli but not for stream water E. coli. Additionally, E. coli surviving in the same habitat tended to have significantly larger (GTG)5 genome similarity. After accounting for the intrinsic impact from the genome, environmental habitat was determined to be a factor influencing some cell surface properties, such as hydrophobicity. The diversity of cell properties and its resulting impact on particle interactions should be considered for environmental fate and transport modeling of aquatic indicator organisms such as E. coli.

8.
Biotechnol Bioeng ; 109(5): 1239-47, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22125231

RESUMO

To establish a production platform for recombinant proteins in rice suspension cells, we first constructed a Gateway-compatible binary T-DNA destination vector. It provided a reliable and effective method for the rapid directional cloning of target genes into plant cells through Agrobacterium-mediated transformation. We used the approach to produce mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) in a rice suspension cell system. The promoter for the αAmy3 amylase gene, which is induced strongly by sugar depletion, drove the expression of mGM-CSF. The resulting recombinant protein was fused with the αAmy3 signal peptide and was secreted into the culture medium. The production of rice-derived mGM-CSF (rmGM-CSF) was scaled up successfully in a 2-L bioreactor, in which the highest yield of rmGM-CSF was 24.6 mg/L. Due to post-translational glycosylation, the molecular weight of rmGM-CSF was larger than that of recombinant mGM-CSF produced in Escherichia coli. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60.


Assuntos
Reatores Biológicos , Biotecnologia/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Oryza/metabolismo , Plantas Geneticamente Modificadas , Agrobacterium/genética , Animais , Técnicas de Cultura de Células , Meios de Cultura/química , DNA Bacteriano , Escherichia coli/genética , Vetores Genéticos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/química , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Camundongos , Peso Molecular , Oryza/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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