scholarly journals RNA-CLAMP Enables Photo-activated Control of CRISPR-Cas9 Gene Editing by Site-specific Intramolecular Cross-linking of the sgRNA

2021 ◽  
Author(s):  
Dongyang Zhang ◽  
Shuaijiang Jin ◽  
Luping Liu ◽  
Ember Tota ◽  
Zijie Li ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Mammalian Cells ◽  
Gene Editing ◽  
Cross Linking ◽  
Stimuli Responsive ◽  
Spatiotemporal Resolution ◽  
Site Specific ◽  
Guide Rna ◽  
Dna Cleavage Activity ◽  
Versatile Tool

AbstractHere we introduce RNA-CLAMP, a technology which enables site-specific and enzymatic cross-linking (clamping) of two selected stem loops within an RNA of interest. Intramolecular clamping of the RNA can disrupt normal RNA function, whereas subsequent photo-cleavage of the crosslinker restores activity. We applied the RNA-CLAMP technique to the single guide RNA of the CRISPR-Cas9 gene editing system. By clamping two stem loops of the single-guide RNA (sgRNA) with a photo-cleavable cross-linker, gene editing was completely silenced. Visible light irradiation cleaved the crosslinker and restored gene editing with high spatiotemporal resolution. Furthermore, by designing two photo-cleavable linkers which are responsive to different wavelength of lights, we achieved multiplexed photo-activation of gene editing in mammalian cells. Notably, although the Cas9-sgRNA RNP is not capable of DNA cleavage activity upon clamping, it maintained the capability to bind to the target DNA. The RNA-CLAMP enabled photo-activated CRISPR-Cas9 gene editing platform offers clean background, free choice of activation wavelength and multiplexing capability. We believe that this technology to precisely and rapidly control gene editing will serve as a versatile tool in the future development of stimuli responsive gene editing technologies. Beyond gene editing, RNA-CLAMP provides a site-specific tool for manipulating the internal structure of functional RNAs.

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 Repair of a Site-Specific DNA Cleavage: Old-School Lessons for Cas9-Mediated Gene Editing

2017 ◽  
Vol 13 (2) ◽  
pp. 397-405 ◽  
Author(s):  
Danielle N. Gallagher ◽  
James E. Haber
Keyword(s):  
Dna Cleavage ◽  
Gene Editing ◽  
Site Specific ◽  
A Site

scholarly journals Chemical Synthesis of Stimuli-Responsive Guide RNA for Conditional Control of CRISPR-Cas9 Gene Editing

2021 ◽  
Author(s):  
Chunmei Gu ◽  
Lu Xiao ◽  
Jiachen Shang ◽  
Xiao Xu ◽  
Luo He ◽  
...  
Keyword(s):  
Chemical Synthesis ◽  
Nucleic Acids ◽  
Gene Editing ◽  
Live Cells ◽  
Stimuli Responsive ◽  
Guide Rna ◽  
Conditional Control ◽  
Target Effects

CRISPR-Cas9 promotes changes in identity or abundance of nucleic acids in live cells and is a programmable modality of broad biotechnological and therapeutic interest. To reduce off-target effects, tools for...

scholarly journals Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA

2015 ◽  
Vol 169 (2) ◽  
pp. 931-945 ◽  
Author(s):  
Sergei Svitashev ◽  
Joshua K. Young ◽  
Christine Schwartz ◽  
Huirong Gao ◽  
S. Carl Falco ◽  
...  
Keyword(s):  
Gene Editing ◽  
Targeted Mutagenesis ◽  
Specific Gene ◽  
Site Specific ◽  
Guide Rna ◽  
Gene Insertion

scholarly journals Enhanced Cas12a multi-gene regulation using a CRISPR array separator

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jens P Magnusson ◽  
Antonio Ray Rios ◽  
Lingling Wu ◽  
Lei S Qi
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Gc Content ◽  
Guide Rna ◽  
Naturally Occurring ◽  
Crispr Array ◽  
Endogenous Genes ◽  
Simultaneous Activation ◽  
Type V ◽  
Array Performance

The type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple guide RNA (gRNAs). Here, we report that Cas12a array performance is hypersensitive to the GC content of gRNA spacers, as high-GC spacers can impair activity of the downstream gRNA. We analyze naturally occurring CRISPR arrays and observe that natural repeats always contain an AT-rich fragment that separates gRNAs, which we term a CRISPR separator. Inspired by this observation, we design short, AT-rich synthetic separators (synSeparators) that successfully remove the disruptive effects between gRNAs. We further demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously underexplored feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.

scholarly journals DNA Binding Induces a cis to trans Switch in Cre Recombinase to Enable Intasome Assembly

2020 ◽  
Author(s):  
Aparna Unnikrishnan ◽  
Carlos D. Amero ◽  
Deepak Kumar Yadav ◽  
Kye Stachowski ◽  
Devante Potter ◽  
...  
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Dna Cleavage ◽  
Gene Editing ◽  
Dna Recombination ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
Dna Cleavage Activity ◽  
Binding Surface ◽  
Synaptic Complexes

ABSTRACTMechanistic understanding of DNA recombination in the Cre-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes. Those studies have suggested a role for protein conformational dynamics that has not been well characterized at the atomic level. We used solution NMR to discover the link between intrinsic flexibility and function in Cre recombinase. TROSY-NMR spectra show the N-terminal and C-terminal catalytic domains (CreNTD, CreCat) to be structurally independent. Amide 15N relaxation measurements of the CreCat domain reveal fast time scale dynamics in most regions that exhibit conformational differences in active and inactive Cre protomers in crystallographic tetramers. However, the C-terminal helix αN, implicated in assembly of synaptic complexes and regulation of DNA cleavage activity via trans protein-protein interactions, is unexpectedly rigid in free Cre. Chemical shift perturbations and intra- and inter-molecular paramagnetic relaxation enhancement (PRE) NMR data reveal an alternative auto-inhibitory conformation for the αN region of free Cre, wherein it packs in cis over the protein DNA binding surface and active site. Moreover, binding to loxP DNA induces a conformational change that dislodge the C-terminus, resulting in a cis to trans switch that is likely to enable protein-protein interactions required for assembly of recombinogenic Cre intasomes. These findings necessitate a re-examination of the mechanisms by which this widely-utilized gene editing tool selects target sites, avoids spurious DNA cleavage activity, and controls DNA recombination efficiency.SIGNIFICANCE STATEMENTThe Cre-loxP system is a widely used gene editing tool that has enabled transformative advances in immunology, neuroscience and cardiovascular research. Still, off-target activities confound research results and present obstacles to biomedical applications. Overcoming those limitations requires understanding the steps leading to assembly of recombination complexes, intasomes. We measured the magnetic properties of nitrogen nuclei in the backbone of the enzyme to correlate its intrinsic dynamics with its function in DNA recognition and cleavage. Remarkably, we found that in the absence of DNA the C-terminus of Cre appears to block the DNA binding surface and active site of the enzyme. Binding to loxP DNA induces a conformational switch that would enable the intermolecular protein-protein interactions required for assembly of recombinogenic Cre intasomes.

scholarly journals Detection of RAG Protein-V(D)J Recombination Signal Interactions Near the Site of DNA Cleavage by UV Cross-Linking

1999 ◽  
Vol 19 (5) ◽  
pp. 3788-3797 ◽  
Author(s):  
Quinn M. Eastman ◽  
Isabelle J. Villey ◽  
David G. Schatz
Keyword(s):  
Active Site ◽  
Dna Cleavage ◽  
Cross Linking ◽  
Specific Interactions ◽  
Cross Link ◽  
Site Specific ◽  
Recombination Signal ◽  
Recombination Signal Sequences ◽  
Close Proximity ◽  
Rag Proteins

ABSTRACT V(D)J recombination is initiated by double-strand cleavage at recombination signal sequences (RSSs). DNA cleavage is mediated by the RAG1 and RAG2 proteins. Recent experiments describing RAG protein-RSS complexes, while defining the interaction of RAG1 with the nonamer, have not assigned contacts immediately adjacent to the site of DNA cleavage to either RAG polypeptide. Here we use UV cross-linking to define sequence- and site-specific interactions between RAG1 protein and both the heptamer element of the RSS and the coding flank DNA. Hence, RAG1-DNA contacts span the site of cleavage. We also detect cross-linking of RAG2 protein to some of the same nucleotides that cross-link to RAG1, indicating that, in the binding complex, both RAG proteins are in close proximity to the site of cleavage. These results suggest how the heptamer element, the recognition surface essential for DNA cleavage, is recognized by the RAG proteins and have implications for the stoichiometry and active site organization of the RAG1-RAG2-RSS complex.

scholarly journals Kinetics of Nuclear Uptake and Site-Specific DNA Cleavage during CRISPR-Directed Gene Editing in Solid Tumor Cells

2020 ◽  
Vol 18 (6) ◽  
pp. 891-902 ◽  
Author(s):  
Kelly Banas ◽  
Natalia Rivera-Torres ◽  
Pawel Bialk ◽  
Byung-Chun Yoo ◽  
Eric B. Kmiec
Keyword(s):  
Tumor Cells ◽  
Solid Tumor ◽  
Dna Cleavage ◽  
Gene Editing ◽  
Site Specific ◽  
Nuclear Uptake ◽  
Kinetics Of

scholarly journals Disabling Cas9 by an anti-CRISPR DNA mimic

2017 ◽  
Author(s):  
Jiyung Shing ◽  
Fuguo Jiang ◽  
Jun-Jie Liu ◽  
Nicholas L. Bray ◽  
Benjamin J. Rauch ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Adaptive Immune System ◽  
Binding Mode ◽  
Human Cells ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
Cas9 Protein ◽  
Potential Applications ◽  
Natural Inhibitors

CRISPR-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known and the potential applications for Cas9 inhibitor proteins in mammalian cells has not fully been established. We show here that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-EM structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif (PAM). Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on pre-formed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.

scholarly journals An educational module to explore CRISPR technologies with a cell-free transcription-translation system

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Daphne Collias ◽  
Ryan Marshall ◽  
Scott P Collins ◽  
Chase L Beisel ◽  
Vincent Noireaux
Keyword(s):  
Undergraduate Students ◽  
Dna Cleavage ◽  
Mammalian Cells ◽  
Genetic Diseases ◽  
Cellular Systems ◽  
Translation System ◽  
Dna Cleavage Activity ◽  
Rna Backbone ◽  
A Cell ◽  
Educational Modules

Abstract Within the last 6 years, CRISPR-Cas systems have transitioned from adaptive defense systems in bacteria and archaea to revolutionary genome-editing tools. The resulting CRISPR technologies have driven innovations for treating genetic diseases and eradicating human pests while raising societal questions about gene editing in human germline cells as well as crop plants. Bringing CRISPR into the classroom therefore offers a means to expose students to cutting edge technologies and to promote discussions about ethical questions at the intersection of science and society. However, working with these technologies in a classroom setting has been difficult because typical experiments rely on cellular systems such as bacteria or mammalian cells. We recently reported the use of an E. coli cell-free transcription-translation (TXTL) system that simplifies the demonstration and testing of CRISPR technologies with shorter experiments and limited equipment. Here, we describe three educational modules intended to expose undergraduate students to CRISPR technologies using TXTL. The three sequential modules comprise (i) designing the RNAs that guide DNA targeting, (ii) measuring DNA cleavage activity in TXTL and (iii) testing how mutations to the targeting sequence or RNA backbone impact DNA binding and cleavage. The modules include detailed protocols, questions for group discussions or individual evaluation, and lecture slides to introduce CRISPR and TXTL. We expect these modules to allow students to experience the power and promise of CRISPR technologies in the classroom and to engage with their instructor and peers about the opportunities and potential risks for society.

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