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 A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9

2017 ◽  
Author(s):  
Yavuz S. Dagdas ◽  
Janice S. Chen ◽  
Samuel H. Sternberg ◽  
Jennifer A. Doudna ◽  
Ahmet Yildiz
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Divalent Cations ◽  
Guide Rna ◽  
Single Molecule Fret ◽  
Target Sites ◽  
Multiple Conformations ◽  
Dna Binding And Cleavage

AbstractThe Cas9 endonuclease is widely utilized for genome engineering applications by programming its single-guide RNA and ongoing work is aimed at improving the accuracy and efficiency of DNA targeting. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule FRET, we identified an intermediate state of S. pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking into its active state. HNH docking requires divalent cations, but not strand scission, and this docked conformation persists following DNA cleavage. Sequence mismatches between the DNA target and guide RNA prevent transitions from the checkpoint intermediate to the active conformation, providing selective avoidance of DNA cleavage at stably bound off-target sites.

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 Heterologous Cas9 and non-homologous end joining as a Potentially Organism-Agnostic Knockout (POAK) system in bacteria

2019 ◽  
Author(s):  
Isaac N Plant
Keyword(s):  
Dna Cleavage ◽  
Sugar Metabolism ◽  
End Joining ◽  
Gene Deletions ◽  
Transformation Protocol ◽  
E Coli ◽  
Rapid Sequence ◽  
Error Prone Repair ◽  
Non Homologous End Joining

Making targeted gene deletions is essential for studying organisms, but is difficult in many prokaryotes due to the inefficiency of homologous recombination based methods. Here, I describe an easily modifiable, single-plasmid system that can be used to make rapid, sequence targeted, markerless knockouts in both a Gram-negative and a Gram-positive organism. The system is comprised of targeted DNA cleavage by Cas9 and error-prone repair by Non-Homologous End Joining (NHEJ) proteins. I confirm previous results showing that Cas9 and NHEJ can make knockouts when NHEJ is expressed before Cas9. Then, I show that Cas9 and NHEJ can be used to make knockouts when expressed simultaneously. I term the new method Potentially Organism-Agnostic Knockout (POAK) system and characterize its function in Escherichia coli and Weissella confusa. First, I develop a novel transformation protocol for W. confusa. Next, I show that, as in E. coli, POAK can create knockouts in W. confusa. Characterization of knockout efficiency across galK in both E. coli and W. confusa showed that while all gRNAs are effective in E. coli, only some gRNAs are effective in W. confusa, and cut site position within a gene does not determine knockout efficiency for either organism. I examine the sequences of knockouts in both organisms and show that POAK produces similar edits in both E. coli and W. confusa. Finally, as an example of the importance of being able to make knockouts quickly, I target W. confusa sugar metabolism genes to show that two sugar importers are not necessary for metabolism of their respective sugars. Having demonstrated that simultaneous expression of Cas9 and NHEJ is sufficient for making knockouts in two minimally related bacteria, POAK represents a hopeful avenue for making knockouts in other under-utilized bacteria.

scholarly journals Rational design of a split-Cas9 enzyme complex

2015 ◽  
Vol 112 (10) ◽  
pp. 2984-2989 ◽  
Author(s):  
Addison V. Wright ◽  
Samuel H. Sternberg ◽  
David W. Taylor ◽  
Brett T. Staahl ◽  
Jorge A. Bardales ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Dna Cleavage ◽  
Rational Design ◽  
Genome Engineering ◽  
Guide Rna ◽  
Protein Architecture ◽  
Target Dna ◽  
Molecular Determinants ◽  
Versatile Tool ◽  
Bacterial Immune Systems

Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.

scholarly journals A versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yaojun Tong ◽  
Tue S. Jørgensen ◽  
Christopher M. Whitford ◽  
Tilmann Weber ◽  
Sang Yup Lee
Keyword(s):  
Genome Engineering ◽  
Genetic Manipulation ◽  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
Base Editing ◽  
Guide Rna ◽  
E Coli ◽  
Potential Basis ◽  
Low Efficiency ◽  
Nucleotide Resolution

AbstractCRISPR base editing is a powerful method to engineer bacterial genomes. However, it restricts editing to single-nucleotide substitutions. Here, to address this challenge, we adapt a CRISPR-Prime Editing-based, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes. It can introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli with high fidelity. Notably, under optimal conditions, the efficiency of 1-bp deletions reach up to 40%. Moreover, deletions of up to 97 bp and insertions up to 33 bp were successful with the toolkit in E. coli, however, efficiencies dropped sharply with increased fragment sizes. With a second guide RNA, our toolkit can achieve multiplexed editing albeit with low efficiency. Here we report not only a useful addition to the genome engineering arsenal for E. coli, but also a potential basis for the development of similar toolkits for other bacteria.

scholarly journals Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a)

2018 ◽  
Vol 115 (21) ◽  
pp. 5444-5449 ◽  
Author(s):  
Digvijay Singh ◽  
John Mallon ◽  
Anustup Poddar ◽  
Yanbo Wang ◽  
Ramreddy Tippana ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Fluorescence Analysis ◽  
Dna Interaction ◽  
Target Sequence ◽  
Solution Ph ◽  
Guide Rna ◽  
Product Release ◽  
Time Observation

CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologs. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9. They both bind any DNA in search of protospacer-adjacent motif (PAM) sequences, verify the target sequence directionally from the PAM-proximal end, and rapidly reject any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ∼16 bp for cleavage, Cpf1 requires an ∼17-bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions, and 5′ guanine in guide-RNA differentially affected different Cpf1 orthologs. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

scholarly journals Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1

2017 ◽  
Author(s):  
Digvijay Singh ◽  
John Mallon ◽  
Anustup Poddar ◽  
Yanbo Wang ◽  
Ramreddy Tippana ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Fluorescence Analysis ◽  
Dna Interaction ◽  
Target Sequence ◽  
Solution Ph ◽  
Guide Rna ◽  
Product Release ◽  
Time Observation

CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We have used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologues. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9, they both bind any DNA in search of PAM (protospacer-adjacent motif) sequences, verifies the target sequence directionally from the PAM-proximal end and rapidly rejects any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ~16 bp for cleavage, Cpf1 requires ~ 17 bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions and 5’ guanine in guide-RNA differentially affected different Cpf1 orthologues. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

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

2018 ◽  
Vol 69 (1) ◽  
pp. 146-157.e3 ◽  
Author(s):  
Ryan Marshall ◽  
Colin S. Maxwell ◽  
Scott P. Collins ◽  
Thomas Jacobsen ◽  
Michelle L. Luo ◽  
...  
Keyword(s):  
Coli Cell ◽  
Translation System ◽  
E Coli

scholarly journals Characterization of the all‐ E. coli transcription‐translation system myTXTL by mass spectrometry

2019 ◽  
Vol 33 (11) ◽  
pp. 1036-1048 ◽  
Author(s):  
David Garenne ◽  
Chase L. Beisel ◽  
Vincent Noireaux
Keyword(s):  
Mass Spectrometry ◽  
Translation System ◽  
E Coli

scholarly journals Live-cell single particle tracking of PRC1 reveals a highly dynamic system with low target site occupancy

2020 ◽  
Author(s):  
Miles K. Huseyin ◽  
Robert J. Klose
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
Genome Engineering ◽  
Target Genes ◽  
Embryonic Stem ◽  
Site Occupancy ◽  
Single Particle Tracking ◽  
Gene Repression ◽  
Live Cells ◽  
Chromatin Binding

AbstractPolycomb repressive complex 1 (PRC1) is an essential chromatin-based repressor of gene transcription. However, how PRC1 engages with chromatin to identify its target genes and achieve gene repression remains poorly defined, representing a major hurdle to our understanding of Polycomb system function. Here we use genome engineering and single particle tracking to dissect how PRC1 binds to chromatin in live mouse embryonic stem cells. We reveal that PRC1 is highly dynamic, with only a small fraction stably interacting with chromatin. By integrating subunit-specific dynamics, chromatin binding, and abundance measurements, we discover that PRC1 exhibits surprisingly low occupancy at target sites. Furthermore, we employ perturbation approaches to uncover how specific components of PRC1 define its kinetics and chromatin binding. Together, these discoveries provide a quantitative understanding of chromatin binding by PRC1 in live cells, and suggests that chromatin modification, as opposed to PRC1 complex occupancy, is central to gene repression.

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