scholarly journals ssRNA/DNA-Sensors via Embedded Strand-Displacement Programs in CRISPR/Cas9 Guides

2018 ◽  
Author(s):  
Noah Jakimo ◽  
Pranam Chatterjee ◽  
Joseph M Jacobson
Keyword(s):  
Computational Design ◽  
Kinetic Characterization ◽  
Proof Of Concept ◽  
Dna Sensors ◽  
Strand Displacement ◽  
Cell Type ◽  
Guide Rna ◽  
Dna Profiles ◽  
Rna And Dna ◽  
Trigger Molecule

RNA and DNA profiles can help classify a variety of biological states, including disease, metabolism and cell type. In a proof-of-concept study on novel genetically encodable components for detecting single-stranded oligonucleotides, we engineered switchable CRISPR guide RNA (swigRNA) with Cas9 affinity that is conditional on sensing an oligo trigger molecule (trigRNA or trigDNA). RNA- and DNA-sensing swigRNAs that serve as off-to-on and on-to-off switches for Cas9 cleavage were achieved by computational design of hybridization dependencies in the guide. This study highlights functional swigRNA structures that implement toehold-gated strand-displacement with their triggers, and should merit further engineering and kinetic characterization.

scholarly journals An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

2021 ◽  
Author(s):  
Eugene V. Gasanov ◽  
Justyna Jędrychowska ◽  
Michal Pastor ◽  
Malgorzata Wiweger ◽  
Axel Methner ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Target Site ◽  
Proof Of Concept ◽  
Intervention Site ◽  
End Joining ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

scholarly journals Conditional guide RNA through two intermediate hairpins for programmable CRISPR/Cas9 function: building regulatory connections between endogenous RNA expressions

2020 ◽  
Vol 48 (20) ◽  
pp. 11773-11784
Author(s):  
Jiao Lin ◽  
Yan Liu ◽  
Peidong Lai ◽  
Huixia Ye ◽  
Liang Xu
Keyword(s):  
Nucleic Acid ◽  
Genetic Regulation ◽  
Binding Activity ◽  
Strand Displacement ◽  
Genetic Circuits ◽  
Reporter System ◽  
Gene Expressions ◽  
Guide Rna ◽  
Naturally Occurring ◽  
Novel Approach

Abstract A variety of nanodevices developed for nucleic acid computation provide great opportunities to construct versatile synthetic circuits for manipulation of gene expressions. In our study, by employing a two-hairpin mediated nucleic acid strand displacement as a processing joint for conditional guide RNA, we aim to build artificial connections between naturally occurring RNA expressions through programmable CRISPR/Cas9 function. This two-hairpin joint possesses a sequence-switching machinery, in which a random trigger strand can be processed to release an unconstrained sequence-independent strand and consequently activate the self-inhibitory guide RNA for conditional gene regulation. This intermediate processor was characterized by the fluorescence reporter system and applied for regulation of the CRISPR/Cas9 binding activity. Using plasmids to generate this sequence-switching machinery in situ, we achieved the autonomous genetic regulation of endogenous RNA expressions controlled by other unrelated endogenous RNAs in both E. coli and human cells. Unlike previously reported strand-displacement genetic circuits, this advanced nucleic acid nanomachine provides a novel approach that can establish regulatory connections between naturally occurring endogenous RNAs. In addition to CRISPR systems, we anticipate this two-hairpin machine can serve as a general processing joint for wide applications in the development of other RNA-based genetic circuits.

scholarly journals A Gold Standard, CRISPR/Cas9-Based Complementation Strategy Reliant on 24 Nucleotide Bookmark Sequences

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 458 ◽  
Author(s):  
François M. Seys ◽  
Peter Rowe ◽  
Edward L. Bolt ◽  
Christopher M. Humphreys ◽  
Nigel P. Minton
Keyword(s):  
Gold Standard ◽  
Mutant Allele ◽  
Target Sequence ◽  
Proof Of Concept ◽  
Wild Type ◽  
Guide Rna ◽  
Simple Strategy ◽  
Clostridium Autoethanogenum ◽  
Gene Knockouts

Phenotypic complementation of gene knockouts is an essential step in establishing function. Here, we describe a simple strategy for ‘gold standard’ complementation in which the mutant allele is replaced in situ with a wild type (WT) allele in a procedure that exploits CRISPR/Cas9. The method relies on the prior incorporation of a unique 24 nucleotide (nt) ‘bookmark’ sequence into the mutant allele to act as a guide RNA target during its Cas9-mediated replacement with the WT allele. The bookmark comprises a 23 nt Cas9 target sequence plus an additional nt to ensure the deletion is in-frame. Here, bookmarks are tailored to Streptococcus pyogenes CRISPR/Cas9 but could be designed for any CRISPR/Cas system. For proof of concept, nine bookmarks were tested in Clostridium autoethanogenum. Complementation efficiencies reached 91%. As complemented strains are indistinguishable from their progenitors, concerns over contamination may be satisfied by the incorporation of ‘watermark’ sequences into the complementing genes.

Sorting for secreted molecule production using a biosensor-in-microdroplet approach

2021 ◽  
Vol 118 (36) ◽  
pp. e2106818118
Author(s):  
Emily K. Bowman ◽  
James M. Wagner ◽  
Shuo-Fu Yuan ◽  
Matthew Deaner ◽  
Claire M. Palmer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Cell Type ◽  
Guide Rna ◽  
E Coli ◽  
Ethyl Methyl ◽  
Methyl Sulfonate ◽  
Acid Lactone ◽  
Mix And Match ◽  
Molecule Production

Sorting large libraries of cells for improved small molecule secretion is throughput limited. Here, we combine producer/secretor cell libraries with whole-cell biosensors using a microfluidic-based screening workflow. This approach enables a mix-and-match capability using off-the-shelf biosensors through either coencapsulation or pico-injection. We demonstrate the cell type and library agnostic nature of this workflow by utilizing single-guide RNA, transposon, and ethyl-methyl sulfonate mutagenesis libraries across three distinct microbes (Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica), biosensors from two organisms (E. coli and S. cerevisiae), and three products (triacetic acid lactone, naringenin, and L-DOPA) to identify targets improving production/secretion.

scholarly journals Use of CRISPR/Cas9-mediated disruption of CNS cell type genes to profile transduction of AAV by neonatal intracerebroventricular delivery in mice

Gene Therapy ◽  
2021 ◽  
Author(s):  
Tess Torregrosa ◽  
Sydney Lehman ◽  
Sam Hana ◽  
Galina Marsh ◽  
Shanqin Xu ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Cell Types ◽  
Brain Regions ◽  
Brain Barrier ◽  
Transduction Efficiency ◽  
Cell Type ◽  
Fluorescent Reporter ◽  
Guide Rna ◽  
Blood Brain ◽  
The Central Nervous System

AbstractAdeno-associated virus (AAV) transduction efficiency and tropism are conventionally determined by high expression of a fluorescent reporter gene. Emerging data has suggested that such conventional methods may underestimate AAV transduction for cells in which reporter expression from AAV vectors is undetectable. To explore an alternative method that captures AAV transduction in cells in which low expression of a cargo is sufficient for the intended activity, we sought after CRISPR/Cas9-mediated gene disruption. In this study, we use AAV to deliver CRISPR/guide RNA designed to abolish the genes NeuN, GFAP, or MOG expressed specifically in neurons, astrocytes, or oligodendrocytes respectively in the central nervous system (CNS) of mice. Abrogated expression of these cell-type-specific genes can be measured biochemically in CNS subregions and provides quantitative assessment of AAV transduction in these CNS cell types. By using this method, we compared CNS transduction of AAV9, AAV-PHP.B, and AAV-PHP.eB delivered via intracerebroventricular injection (ICV) in neonatal mice. We found both AAV-PHP.B and AAV-PHP.eB resulted in marked disruption of the NeuN gene by CRISPR/Cas9, significantly greater than AAV9 in several brain regions and spinal cord. In contrast, only modest disruption of the GFAP gene and the MOG gene was observed by all three AAV variants. Since the procedure of ICV circumvents the blood–brain barrier, our data suggests that, independent of their ability to cross the blood–brain barrier, AAV-PHP.B variants also exhibit remarkably improved neuronal transduction in the CNS. We anticipate this approach will facilitate profiling of AAV cellular tropism in murine CNS.

scholarly journals Human MiniPromoters for ocular-rAAV expression in ON bipolar, cone, corneal, endothelial, Müller glial, and PAX6 cells

Gene Therapy ◽  
2021 ◽  
Author(s):  
Andrea J. Korecki ◽  
Jorge L. Cueva-Vargas ◽  
Oriol Fornes ◽  
Jessica Agostinone ◽  
Rachelle A. Farkas ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Adult Mouse ◽  
Corneal Stroma ◽  
Cell Type ◽  
Preclinical Research ◽  
Gene Therapies ◽  
Small Regulatory Rna ◽  
Human Proteins ◽  
Clinical Gene Therapy ◽  
Rna And Dna

AbstractSmall and cell-type restricted promoters are important tools for basic and preclinical research, and clinical delivery of gene therapies. In clinical gene therapy, ophthalmic trials have been leading the field, with over 50% of ocular clinical trials using promoters that restrict expression based on cell type. Here, 19 human DNA MiniPromoters were bioinformatically designed for rAAV, tested by neonatal intravenous delivery in mouse, and successful MiniPromoters went on to be tested by intravitreal, subretinal, intrastromal, and/or intravenous delivery in adult mouse. We present promoter development as an overview for each cell type, but only show results in detail for the recommended MiniPromoters: Ple265 and Ple341 (PCP2) ON bipolar, Ple349 (PDE6H) cone, Ple253 (PITX3) corneal stroma, Ple32 (CLDN5) endothelial cells of the blood–retina barrier, Ple316 (NR2E1) Müller glia, and Ple331 (PAX6) PAX6 positive. Overall, we present a resource of new, redesigned, and improved MiniPromoters for ocular gene therapy that range in size from 784 to 2484 bp, and from weaker, equal, or stronger in strength relative to the ubiquitous control promoter smCBA. All MiniPromoters will be useful for therapies involving small regulatory RNA and DNA, and proteins ranging from 517 to 1084 amino acids, representing 62.9–90.2% of human proteins.

scholarly journals Designing Minimal Genomes Using Whole-Cell Models

2018 ◽  
Author(s):  
Joshua Rees ◽  
Oliver Chalkley ◽  
Sophie Landon ◽  
Oliver Purcell ◽  
Lucia Marucci ◽  
...  
Keyword(s):  
In Silico ◽  
Genome Engineering ◽  
Cell Model ◽  
Mycoplasma Genitalium ◽  
Computational Design ◽  
Functional Assay ◽  
Proof Of Concept ◽  
Computational Tools ◽  
Whole Cell ◽  
Genome Design

AbstractIn the future, entire genomes tailored to specific functions and environments could be designed using computational tools. However, computational tools for genome design are currently scarce. Here we present algorithms that enable the use of design-simulate-test cycles for genome design, using genome minimisation as a proof-of-concept. Minimal genomes are ideal for this purpose as they have a simple functional assay, the cell either replicates or not. We used the first (and currently only published) whole-cell model, for the bacterium Mycoplasma genitalium. Our computational design-simulate-test cycles discovered novel in-silico minimal genomes smaller than JCVI-Syn3.0, a bacteria with, currently, the smallest genome that can be grown in pure culture. In the process, we identified 10 low essentiality genes, 18 high essentiality genes, and produced evidence for at least two Mycoplasma genitalium in-silico minimal genomes. This work brings combined computational and laboratory genome engineering a step closer.

scholarly journals A mechanism for ligand gated strand displacement in ZTP riboswitch transcription regulation

2019 ◽  
Author(s):  
Eric J. Strobel ◽  
Luyi Cheng ◽  
Katherine E. Berman ◽  
Paul D. Carlson ◽  
Julius B. Lucks
Keyword(s):  
Rna Structure ◽  
Clostridium Beijerinckii ◽  
Strand Displacement ◽  
Structural Determinants ◽  
Rna Molecules ◽  
Guide Rna ◽  
Folding Pathways ◽  
Rna Biogenesis ◽  
And Function ◽  
Downstream Gene Expression

AbstractCotranscriptional folding is an obligate step of RNA biogenesis that can guide RNA structure and function by forming transient intermediate folds. This is especially true for transcriptional riboswitches in the which the formation of ligand-dependent structures during transcription regulates downstream gene expression. However, the intermediate structures that comprise cotranscriptional RNA folding pathways and the mechanisms that enable transit between them remain largely unknown. Here we determine the series of cotranscriptional folds and rearrangements that mediate antitermination by the Clostridium beijerinckii pfl riboswitch in response to the purine biosynthetic intermediate ZMP. We uncover sequence and structural determinants that modulate a regulatory RNA strand displacement reaction and identify biases within natural ZTP riboswitch sequences that promote on-pathway folding. Our findings establish a mechanism for ZTP riboswitch antitermination and suggest general strategies by which nascent RNA molecules can navigate cotranscriptional folding pathways efficiently.

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