scholarly journals DNA unwinding is the primary determinant of CRISPR-Cas9 activity

2017 ◽  
Author(s):  
Shanzhong Gong ◽  
Helen Hong Yu ◽  
Kenneth A. Johnson ◽  
David W. Taylor
Keyword(s):  
Genome Engineering ◽  
Nuclease Activity ◽  
Loop Formation ◽  
Base Pairs ◽  
Guide Rna ◽  
Target Strand ◽  
Primary Determinant ◽  
Specificity Constant ◽  
Rate Limiting

SummaryBacterial adaptive immunity utilizes RNA-guided surveillance complexes composed of CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target foreign nucleic acids for destruction. Cas9, a type II CRISPR-Cas effector complex, can be programed with a single guide RNA that base-pairs with the target strand of dsDNA, displacing the non-target strand to create an R-loop, where the HNH and RuvC nuclease domains can cleave opposing strands. Cas9 has been repurposed for a variety of important genome engineering applications. While many structural and biochemical studies have shed light on the mechanism of Cas9 cleavage, a clear unifying model has yet to emerge. Our detailed kinetic characterization of the enzyme reveals that DNA binding is reversible, R-loop formation is rate-limiting, occurring in two steps, one for each of the nuclease domains. Although the HNH nuclease activity is stimulated by Mg2+ with a single measureable Kd, the RuvC activity requires two distinct Mg2+ binding events. The specificity constant for cleavage is determined through an induced-fit mechanism as the product of the equilibrium binding affinity for DNA and the rate of R-loop formation.

scholarly journals Mechanism of R-loop formation and conformational activation of Cas9

2021 ◽  
Author(s):  
Martin Pacesa ◽  
Martin Jinek
Keyword(s):  
Dna Cleavage ◽  
Structural Basis ◽  
Seed Region ◽  
Loop Formation ◽  
Base Pairs ◽  
Guide Rna ◽  
Target Strand ◽  
Target Dna ◽  
Dna Strand ◽  
Dna Substrate

Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications. Despite extensive studies, the precise mechanism of target DNA binding and on-/off-target discrimination remains incompletely understood. Here we report cryo-EM structures of intermediate binding states of Streptococcus pyogenes Cas9 that reveal domain rearrangements induced by R-loop propagation and PAM-distal duplex positioning. At early stages of binding, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the PAM-distal duplex of the DNA substrate. Target hybridisation past the seed region positions the guide-target heteroduplex into the central binding channel and results in a conformational rearrangement of the REC lobe. Extension of the R-loop to 16 base pairs triggers the relocation of the HNH domain towards the target DNA strand in a catalytically incompetent conformation. The structures indicate that incomplete target strand pairing fails to induce the conformational displacements necessary for nuclease domain activation. Our results establish a structural basis for target DNA-dependent activation of Cas9 that advances our understanding of its off-target activity and will facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.

scholarly journals Rapid and scalable characterization of CRISPR technologies using an E. coli cell-free transcription-translation system

2017 ◽  
Author(s):  
Ryan Marshall ◽  
Colin S. Maxwell ◽  
Scott P. Collins ◽  
Michelle L. Luo ◽  
Thomas Jacobsen ◽  
...  
Keyword(s):  
Protein Purification ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Gene Repression ◽  
Live Cells ◽  
Translation System ◽  
Guide Rna ◽  
E Coli ◽  
Translation Systems

ABSTRACTCRISPR-Cas systems have offered versatile technologies for genome engineering, yet their implementation has been outpaced by the ongoing discovery of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation systems (TXTL) to vastly improve the speed and scalability of CRISPR characterization and validation. Unlike prior approaches that require protein purification or live cells, TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression. To demonstrate the applicability of TXTL, we rapidly measure guide RNA-dependent DNA cleavage and gene repression for single- and multi-effector CRISPR-Cas systems, accurately predict the strength of gene repression in E. coli, quantify the inhibitory activity of anti-CRISPR proteins, and develop a fast and scalable high-throughput screen for protospacer-adjacent motifs. These examples underscore the potential of TXTL to facilitate the characterization and application of CRISPR technologies across their many uses.

scholarly journals Unified energetics analysis unravels SpCas9 cleavage activity for optimal gRNA design

2019 ◽  
Vol 116 (18) ◽  
pp. 8693-8698 ◽  
Author(s):  
Dong Zhang ◽  
Travis Hurst ◽  
Dongsheng Duan ◽  
Shi-Jie Chen
Keyword(s):  
Genome Engineering ◽  
Learning Approaches ◽  
Loop Formation ◽  
Unified Framework ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cleavage Activity ◽  
Protospacer Adjacent Motif ◽  
Traditional Approaches ◽  
Target Effects

While CRISPR/Cas9 is a powerful tool in genome engineering, the on-target activity and off-target effects of the system widely vary because of the differences in guide RNA (gRNA) sequences and genomic environments. Traditional approaches rely on separate models and parameters to treat on- and off-target cleavage activities. Here, we demonstrate that a free-energy scheme dominates the Cas9 editing efficacy and delineate a method that simultaneously considers on-target activities and off-target effects. While data-driven machine-learning approaches learn rules to model particular datasets, they may not be as transferrable to new systems or capable of producing new mechanistic insights as principled physical approaches. By integrating the energetics of R-loop formation under Cas9 binding, the effect of the protospacer adjacent motif sequence, and the folding stability of the whole single guide RNA, we devised a unified, physical model that can apply to any cleavage-activity dataset. This unified framework improves predictions for both on-target activities and off-target efficiencies of spCas9 and may be readily transferred to other systems with different guide RNAs or Cas9 ortholog proteins.

scholarly journals Organelle Genetics in Plants

2021 ◽  
Vol 22 (4) ◽  
pp. 2104
Author(s):  
Pedro Robles ◽  
Víctor Quesada
Keyword(s):  
Rna Editing ◽  
Posttranscriptional Regulation ◽  
Genome Engineering ◽  
Plant Mitochondria ◽  
Plastid Dna ◽  
Chloroplast Genomes ◽  
Wide Range ◽  
Organelle Genetics ◽  
Selection Of

Eleven published articles (4 reviews, 7 research papers) are collected in the Special Issue entitled “Organelle Genetics in Plants.” This selection of papers covers a wide range of topics related to chloroplasts and plant mitochondria research: (i) organellar gene expression (OGE) and, more specifically, chloroplast RNA editing in soybean, mitochondria RNA editing, and intron splicing in soybean during nodulation, as well as the study of the roles of transcriptional and posttranscriptional regulation of OGE in plant adaptation to environmental stress; (ii) analysis of the nuclear integrants of mitochondrial DNA (NUMTs) or plastid DNA (NUPTs); (iii) sequencing and characterization of mitochondrial and chloroplast genomes; (iv) recent advances in plastid genome engineering. Here we summarize the main findings of these works, which represent the latest research on the genetics, genomics, and biotechnology of chloroplasts and mitochondria.

scholarly journals Inhibition of CRISPR-Cas12a DNA targeting by nucleosomes and chromatin

2021 ◽  
Vol 7 (11) ◽  
pp. eabd6030
Author(s):  
Isabel Strohkendl ◽  
Fatema A. Saifuddin ◽  
Bryan A. Gibson ◽  
Michael K. Rosen ◽  
Rick Russell ◽  
...  
Keyword(s):  
Histone Modifications ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Eukaryotic Cells ◽  
Loop Formation ◽  
Chromatin Compaction ◽  
Dna Targeting ◽  
Genomic Regions ◽  
Dna Bendability ◽  
Nucleosome Arrays

Genome engineering nucleases must access chromatinized DNA. Here, we investigate how AsCas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates target accessibility to Cas12a and determines the extent to which both steps of binding—PAM recognition and R-loop formation—are inhibited by the nucleosome. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing target-proximal dCas9 enhances DNA cleavage rates over 10-fold. Unexpectedly, Cas12a readily cleaves internucleosomal linker DNA within chromatin-like, phase-separated nucleosome arrays. DNA targeting is reduced only ~5-fold due to neighboring nucleosomes and chromatin compaction. This work explains the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted genomic regions in cells. We conclude that nucleosome unwrapping regulates accessibility to CRISPR-Cas nucleases and propose that increasing nucleosome breathing dynamics will improve DNA targeting in eukaryotic cells.

scholarly journals Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction

2021 ◽  
Vol 22 (2) ◽  
pp. 734
Author(s):  
Paul K. Varghese ◽  
Mones Abu-Asab ◽  
Emilios K. Dimitriadis ◽  
Monika B. Dolinska ◽  
George P. Morcos ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Large Scale ◽  
Magnetic Beads ◽  
Enzymatic Reaction ◽  
Oculocutaneous Albinism ◽  
Transmission Electron ◽  
Hill Kinetics ◽  
Rate Limiting ◽  
Human Tyrosinase

Human Tyrosinase (Tyr) is the rate-limiting enzyme of the melanogenesis pathway. Tyr catalyzes the oxidation of the substrate L-DOPA into dopachrome and melanin. Currently, the characterization of dopachrome-related products is difficult due to the absence of a simple way to partition dopachrome from protein fraction. Here, we immobilize catalytically pure recombinant human Tyr domain (residues 19–469) containing 6xHis tag to Ni-loaded magnetic beads (MB). Transmission electron microscopy revealed Tyr-MB were within limits of 168.2 ± 24.4 nm while the dark-brown melanin images showed single and polymerized melanin with a diameter of 121.4 ± 18.1 nm. Using Hill kinetics, we show that Tyr-MB has a catalytic activity similar to that of intact Tyr. The diphenol oxidase reactions of L-DOPA show an increase of dopachrome formation with the number of MB and with temperature. At 50 °C, Tyr-MB shows some residual catalytic activity suggesting that the immobilized Tyr has increased protein stability. In contrast, under 37 °C, the dopachrome product, which is isolated from Tyr-MB particles, shows that dopachrome has an orange-brown color that is different from the color of the mixture of L-DOPA, Tyr, and dopachrome. In the future, Tyr-MB could be used for large-scale productions of dopachrome and melanin-related products and finding a treatment for oculocutaneous albinism-inherited diseases.

scholarly journals Characterization of a Novel Thermobifida fusca Bacteriophage P318

Viruses ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1042
Author(s):  
Cheepudom ◽  
Lin ◽  
Lee ◽  
Meng
Keyword(s):  
Plant Cell Wall ◽  
Hydrolytic Enzymes ◽  
Thermobifida Fusca ◽  
Genome Replication ◽  
Putative Orfs ◽  
Base Pairs ◽  
Double Stranded Dna ◽  
Virion Morphogenesis ◽  
Genome Information

Thermobifida fusca is of biotechnological interest due to its ability to produce an array of plant cell wall hydrolytic enzymes. Nonetheless, only one T. fusca bacteriophage with genome information has been reported to date. This study was aimed at discovering more relevant bacteriophages to expand the existing knowledge of phage diversity for this host species. With this end in view, a thermostable T. fusca bacteriophage P318, which belongs to the Siphoviridae family, was isolated and characterized. P318 has a double-stranded DNA genome of 48,045 base pairs with 3′-extended COS ends, on which 52 putative ORFs are organized into clusters responsible for the order of genome replication, virion morphogenesis, and the regulation of the lytic/lysogenic cycle. In comparison with T. fusca and the previously discovered bacteriophage P1312, P318 has a much lower G+C content in its genome except at the region encompassing ORF42, which produced a protein with unknown function. P1312 and P318 share very few similarities in their genomes except for the regions encompassing ORF42 of P318 and ORF51 of P1312 that are homologous. Thus, acquisition of ORF42 by lateral gene transfer might be an important step in the evolution of P318.

scholarly journals Molecular characterization of a beta zero-thalassemia resulting from a 1.4 kilobase deletion

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 636-641 ◽  
Author(s):  
R Anand ◽  
CD Boehm ◽  
HH Jr Kazazian ◽  
EF Vanin
Keyword(s):  
Transcriptional Start Site ◽  
Globin Gene ◽  
Event Analysis ◽  
Beta Globin ◽  
Base Pairs ◽  
Dna Polymerase Alpha ◽  
Beta Globin Gene ◽  
American Black ◽  
The Individual

Abstract We report the characterization of a beta zero-thalassemia in an American Black with unusually high HbA2 and HbF levels. Genomic southern analysis indicated that the individual was heterozygous for a deletion that began within the second intervening sequence of the beta- globin gene and extended approximately 1.4 kb in the 5′ direction. A clone spanning the breakpoint on the abnormal chromosome was isolated and further mapped, and the deletion joint was sequenced. Comparison of the normal beta-globin gene and its 5′ flanking region with the deletion joint sequence indicated that the 5′ breakpoint for this deletion was 484 base pairs (bp) 5′ to the transcriptional start site for the beta-globin gene and the 3′ breakpoint was 908 bp into the beta- globin gene; the deletion removed a total of 1,393 bp. Comparison of the normal 5′ and 3′ breakpoint sequences indicated that this deletion was the result of a “clean” nonhomologous breakage and reunion event; ie, no spurious bases were added during the recombinational event. Analysis of the breakpoints of this deletion together with the breakpoints of two other small deletions involving the beta-globin gene suggests that the breakpoints may occur at DNA polymerase alpha pause sites.

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