scholarly journals Repressing Integrase attachment site operation with CRISPR-Cas9 in E. coli

2017 ◽  
Author(s):  
Andrey Shur ◽  
Richard M. Murray
Keyword(s):  
Dna Sequences ◽  
Dna Recombination ◽  
Attachment Site ◽  
Memory Systems ◽  
Rna Sequences ◽  
Guide Rna ◽  
Future Goals ◽  
Attachment Sites ◽  
Synthetic Cell ◽  
Specific Pair

AbstractSerine integrases are bacteriophage proteins responsible for integrating the phage genome into that of the host. Synthetic biologists have co-opted these proteins into useful tools for permanent DNA logic, utilizing their specific DNA recombination abilities to build synthetic cell differentiation and genetic memory systems. Each integrase has a specific pair of DNA sequences (attP/attB sites) that it recombines, but multiple identical sites can result in unpredictable recombination. We have developed a way to control integrase activity on identical attP/attB sites by using catalytically dead Cas9 (dCas9) as a programmable binding protein that can compete with integrase for binding to specific attachment sites. Utilizing a plasmid that contains two identical Bxb1 attP sites, integration can be repressed up to 8 fold at either one of the two attP sites when guide RNA and dCas9 are present. Guide RNA sequences that bind specifically to attB, or either of two attP sites, have been developed. Future goals are to utilize this technology to construct larger and more complex integrase logic circuits.

scholarly journals Tn7-CRISPR-Cas12K elements manage pathway choice using truncated repeat-spacer units to target tRNA attachment sites

2021 ◽  
Author(s):  
Shan-Chi Hsieh ◽  
Joseph E. Peters
Keyword(s):  
Attachment Site ◽  
Mobile Elements ◽  
Information Support ◽  
Trna Genes ◽  
Type I ◽  
Guide Rna ◽  
Guide Rnas ◽  
Attachment Sites ◽  
Type V ◽  
Future Work

AbstractCRISPR-Cas systems provide a defense against mobile elements. These defense systems have been naturally coopted multiple times for guide RNA-directed transposition by Tn7-like transposons. Elements associated with a type I-F CRISPR-Cas system categorize guide RNAs, maintaining a standard CRISPR array capable of acquiring new spacers targeting other mobile elements while maintaining a special guide RNA allowing integration into a conserved site in the chromosome called an attachment site. We show here that Tn7-like elements associated with a type V-K (Cas12K-based) system use a similar strategy to target diverse tRNA genes as attachment sites. These guides are encoded as truncated minimal repeat-spacer units and are found in distinct locations. Multiple pieces of information support that V-K guide RNAs are acquired using a type I-D adaptation system, but remain private to the V-K transposition process. This catalog of Cas12K elements and naturally occurring insertions will help future work engineering precision integration systems.

DESIGNED SELF-ORGANIZATION FOR MOLECULAR OPTOELECTRONIC SENSORS

2007 ◽  
Vol 17 (02) ◽  
pp. 311-326 ◽  
Author(s):  
Michael Norton
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
Terahertz Spectroscopy ◽  
Attachment Site ◽  
Surface Immobilization ◽  
Surface Attachment ◽  
One Dimensional ◽  
Synthetic Dna ◽  
Attachment Sites ◽  
Sensitivity And Selectivity

The convergence of terahertz spectroscopy and single molecule experimentation offers significant promise of enhancement in sensitivity and selectivity in molecular recognition, identification and quantitation germane to military and security applications. This paper provides a brief overview of the constraints set by single molecule recognition systems and reports the results of experiments which address fundamental barriers to the integration of large, patterned bio-compatible molecular opto-electronic systems with silicon based microelectronic systems. Central to this thrust is an approach involving sequential epitaxy on surface bound single stranded DNA one-dimensional substrates. The challenge of producing highly structured macromolecular substrates, which are necessary in order to implement molecular nanolithography, has been addressed experimentally by combining “designer” synthetic DNA with biosynthetically derived plasmid components. By design, these one dimensional templates are composed of domains which contain sites which are recognized, and therefore addressable by either complementary DNA sequences and/or selected enzymes. Such design is necessary in order to access the nominal 2 nm linewidth potential resolution of nanolithography on these one-dimensional substrates. The recognition and binding properties of DNA ensure that the lithographic process is intrinsically self-organizing, and therefore self-aligning, a necessity for assembly processes at the requisite resolution. Another requirement of this molecular epitaxy approach is that the substrate must be immobilized. The challenge of robust surface immobilization is being addressed via the production of the equivalent of molecular tube sockets. In this application, multi-valent core-shell fluorescent quantum dots provide a mechanism to prepare surface attachment sites with a pre-determined 1:1 attachment site : substrate (DNA) molecule ratio.

scholarly journals Massively parallel kinetic profiling of natural and engineered CRISPR nucleases

2019 ◽  
Author(s):  
Stephen K. Jones ◽  
John A. Hawkins ◽  
Nicole V. Johnson ◽  
Cheulhee Jung ◽  
Kuang Hu ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Massively Parallel ◽  
Biophysical Model ◽  
Rna Sequences ◽  
Time Resolved ◽  
Guide Rna ◽  
Dna Library ◽  
Cleavage Specificity ◽  
Target Binding

AbstractEngineered Streptococcus pyogenes (Sp) Cas9s and Acidaminococcus sp. (As) Cas12a (formerly Cpf1) improve cleavage specificity in human cells. However, the fidelity, enzymatic mechanisms, and cleavage products of emerging CRISPR nucleases have not been profiled systematically across partially mispaired off-target DNA sequences. Here, we describe NucleaSeq— nuclease digestion and deep sequencing—a massively parallel platform that measures cleavage kinetics and captures the time-resolved identities of cleaved products for more than ten thousand DNA targets that include mismatches, insertions, and deletions relative to the guide RNA. The binding specificity of each enzyme is measured on the same DNA library via the chip-hybridized association mapping platform (CHAMP). Using this integrated cleavage and binding platform, we profile four SpCas9 variants and AsCas12a. Engineered Cas9s retain wtCas9-like off-target binding but increase cleavage specificity; Cas9-HF1 shows the most dramatic increase in cleavage specificity. Surprisingly, wtCas12a—reported as a more specific nuclease in cells—has cleavage specificity similar to wtCas9 in vitro. Initial cleavage position and subsequent end-trimming vary across nucleases, guide RNA sequences, and position and base identity of mispairs in target DNAs. Using these large datasets, we develop a biophysical model that reveals mechanistic insights into off-target cleavage activities by these nucleases. More broadly, NucleaSeq enables rapid, quantitative, and systematic comparison of the specificities and cleavage products of engineered and natural nucleases.

Differential dissociation of chromatin digests: a novel approach revealing a hierarchy of DNA-protein interactions within chromatin domains

1991 ◽  
Vol 99 (3) ◽  
pp. 503-513
Author(s):  
A.V. Lichtenstein ◽  
M.M. Zaboikin ◽  
N.I. Sjakste ◽  
R.P. Alechina
Keyword(s):  
Protein Interactions ◽  
Dna Sequences ◽  
Attachment Site ◽  
Chromatin Domain ◽  
Dna Fragments ◽  
S1 Nuclease ◽  
Novel Approach ◽  
Attachment Sites ◽  
Dna Protein Interactions

We describe here a novel approach to the dissection of chromatin structure by extracting DNA fragments from digested nuclei irreversibly immobilized (via proteins) on Celite columns. Three successive gradients (NaCl, LiCl-urea, temperature) are used to release three families of DNA fragments: namely, the ‘DNA adherence’ classes DNA-0, DNA-I and DNA-II, respectively. This ‘protein image’ DNA chromatography separates DNA fragments in accordance with the tightness of their bonds with proteins in situ. There are at least two DNA-skeleton attachment sites differing from each other by their resistance to the dissociating agents used as well as their susceptibility to DNAase I and S1 nuclease treatments, DNA cross-linking and single-stranded breaks. Several lines of evidence show a specific, topological rather than chemical, DNA-protein linkage at the tight attachment site. A hierarchy of chromatin loops demarcated by these attachment sites was determined. The technique described is generally applicable and can be used both to probe DNA-protein interactions and to map specific DNA sequences within the chromatin domain.

scholarly journals Proof of concept continuous event logging in living cells

2017 ◽  
Author(s):  
Andrey Shur ◽  
Richard M. Murray
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Dna Sequences ◽  
Rna Binding ◽  
Attachment Site ◽  
Genetic Circuit ◽  
Genetic Changes ◽  
Guide Rna ◽  
Molecular Events ◽  
Long Read

AbstractCells must detect and respond to molecular events such as the presence or absence of specific small molecules. To accomplish this, cells have evolved methods to measure the presence and concentration of these small molecules in their environment and enact changes in gene expression or behavior. However, cells don’t usually change their DNA in response to such outside stimuli. In this work, we have engineered a genetic circuit that can enact specific and controlled genetic changes in response to changing small molecule concentrations. Known DNA sequences can be repeatedly integrated into a genomic array such that their identity and order encodes information about past small molecule concentrations that the cell has experienced. To accomplish this, we use catalytically inactive CRISPR-Cas9 (dCas9) to bind to and block attachment sites for the integrase Bxb1. Therefore, through the co-expression of dCas9 and guide RNA, Bxb1 can be directed to integrate one of two engineered plasmids, which correspond to two orthogonal small molecule inducers that can be recorded with this system. We identified the optimal location of guide RNA binding to the Bxb1 attP integrase attachment site, and characterized the detection limits of the system by measuring the minimal small molecule concentration and shortest induction time necessary to produce measurable differences in array composition as read out by Oxford Nanopore long read sequencing technology.

scholarly journals The effect of attachment site mutations on strand exchange in bacteriophage lambda site-specific recombination.

Genetics ◽  
1989 ◽  
Vol 122 (4) ◽  
pp. 727-736
Author(s):  
C E Bauer ◽  
J F Gardner ◽  
R I Gumport ◽  
R A Weisberg
Keyword(s):  
Attachment Site ◽  
Strand Exchange ◽  
Segregation Pattern ◽  
Base Substitution ◽  
Base Pairs ◽  
Multiple Mutation ◽  
Attachment Sites ◽  
Dna Strands ◽  
Substitution Mutations ◽  
Overlap Region

Abstract Recombination of phage lambda attachment sites occurs by sequential exchange of the DNA strands at two specific locations. The first exchange produces a Holliday structure, and the second resolves it to recombinant products. Heterology for base substitution mutations in the region between the two strand exchange points (the overlap region) reduces recombination; some mutations inhibit the accumulation of Holliday structures, others inhibit their resolution to recombinant products. To see if heterology also alters the location of the strand exchange points, we determined the segregation pattern of three single and one multiple base pair substitution mutations of the overlap region in crosses with wild type sites. The mutations are known to differ in the severity of their recombination defect and in the stage of strand exchange they affect. The three single mutations behaved similarly: each segregated into both products of recombination, and the two products of a single crossover were frequently nonreciprocal in the overlap region. In contrast, the multiple mutation preferentially segregated into one of the two recombinant products, and the two products of a single crossover appeared to be fully reciprocal. The simplest explanation of the segregation pattern of the single mutations is that strand exchanges occur at the normal locations to produce recombinants with mismatched base pairs that are frequently repaired. The segregation pattern of the multiple mutation is consistent with the view that both strand exchanges usually occur to one side of the mutant site. We suggest that the segregation pattern of a particular mutation is determined by which stage of strand exchange it inhibits and by the severity of the inhibition.

Modified S-transform as a tool to identify secondary structure elements in RNA

2017 ◽  
Vol 13 (4) ◽  
Author(s):  
Nancy Singh ◽  
Sunil Datt Sharma ◽  
Ragothaman M. Yennamalli
Keyword(s):  
Signal Processing ◽  
Secondary Structure ◽  
Dna Sequences ◽  
Tertiary Structure ◽  
Processing Method ◽  
Rna Sequences ◽  
Signal Processing Method ◽  
S Transform ◽  
Highly Correlated ◽  
Signal Processing Methods

AbstractIn this article, we describe the applicability of a signal processing method, specifically the modified S-transform (MST) method, on RNA sequences to identify periodicities between 2 and 11. MicroRNAs (miRNA) are associated with gene regulation and gene silencing and thus have wide applications in biological sciences. Also, the functionality of miRNA is highly associated with its secondary structures (stem, bulge and loop). Signal processing methods have been previously applied on genomic data to reveal the periodicities that determine a wide variety of biological functions, ranging from exon detection to microsatellite identification in DNA sequences. However, there has been less focus on RNA-based signal processing. Here, we show that the signal processing method can be successfully applied to miRNA sequences. We observed that these periodicities are highly correlated with the secondary structure of miRNA and such methods could possibly be used as indicators of secondary and tertiary structure formation.

DNA-RNA complexes that might represent transient attachment sites of nuclear DNA to the matrix

1990 ◽  
Vol 95 (4) ◽  
pp. 667-674
Author(s):  
C. Patriotis ◽  
M. Andreeva ◽  
M. Pascaleva ◽  
V. Ivanov ◽  
L. Djondjurov
Keyword(s):  
Dna Sequences ◽  
Nuclear Dna ◽  
Rna Polymerase Iii ◽  
Erythroid Differentiation ◽  
Rnase H ◽  
Dna And Rna ◽  
Attachment Sites ◽  
The Matrix ◽  
Exo Iii ◽  
Rna Complexes

In this study we describe DNA-RNA complexes in matrix DNA of Friend cells. The presence of such unusual structures is confirmed by the following evidence. When a preparation of matrix DNA is electrophoresed in agarose an RNA component always migrates together with DNA. There should be a close interaction between DNA and RNA in such a preparation because the presence of the RNA component causes resistance of DNA to DNase I and Exo III. An intimate, hybrid-type association of part of the RNA component with DNA is indicated also by the fact that about 20% of this RNA is sensitive to RNase H. By specific inhibition of the RNA synthesis with alpha-amanitin and actinomycin D it was shown that the bulk of associated RNA is transcribed by RNA polymerase III. Hybridization experiments showed similarity between the DNA sequences isolated from the complexes and those from the base of dehistonized DNA loops obtained by high-salt extraction of nuclei. This observation suggests that the complexes might represent attachment sites of nuclear DNA to the matrix: possibly, the attachment is mediated via the RNA component. Experiments with induction of erythroid differentiation indicated that a profound reorganization of the nucleus, accompanying terminal differentiation, leads to a striking reduction in the number of complexes and thus in the number of attachment sites. This suggests that the complexes should function as transient attachment sites.

scholarly journals Enhancement of target specificity of CRISPR–Cas12a by using a chimeric DNA–RNA guide

2020 ◽  
Vol 48 (15) ◽  
pp. 8601-8616 ◽  
Author(s):  
Hanseop Kim ◽  
Wi-jae Lee ◽  
Yeounsun Oh ◽  
Seung-Hun Kang ◽  
Junho K Hur ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Engineering ◽  
Target Genes ◽  
Target Sequence ◽  
Energy Potential ◽  
Guide Rna ◽  
Sequence Recognition ◽  
Protospacer Adjacent Motif ◽  
Effective Manner ◽  
Target Dna

Abstract The CRISPR–Cas9 system is widely used for target-specific genome engineering. CRISPR–Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR–Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR–Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.

AN ANATOMICAL STUDY OF THE LIGAMENTS OF THE ULNAR COMPARTMENT OF THE WRIST

Hand Surgery ◽  
2003 ◽  
Vol 08 (02) ◽  
pp. 219-226 ◽  
Author(s):  
Saburo Sasao ◽  
Moroe Beppu ◽  
Hitoshi Kihara ◽  
Kazuaki Hirata ◽  
Masayuki Takagi
Keyword(s):  
Three Dimensional ◽  
Attachment Site ◽  
Ulnar Collateral Ligament ◽  
Anatomical Study ◽  
Styloid Process ◽  
Dimensional Structure ◽  
Collateral Ligament ◽  
Ulnar Styloid ◽  
Attachment Sites ◽  
Triangular Fibrocartilage

The ligamentous structures of the triangular fibrocartilage complex (TFCC) and their attachments were examined anatomically and histologically using fresh and embalmed cadavers. The TFCC was observed to have a three-dimensional structure consisting of three palmar ligaments — the short radiolunate (SRL), ulnolunate (UL), and ulnotriquetral (UT) ligaments. In addition, the attachment site of the ulnocarpal ligament (UC), which had been previously unknown, was identified. The dorsal components of the TFCC have been previously reported to consist solely of the extensor carpi ulnaris (ECU) subsheath; however, the ligamentous components running from the ulnar styloid process to the triquetrum were found at a layer deeper than the floor of the ECU subsheath. The UC has been reported previously as a two-dimensional structure, but there has been some disagreement as to its attachment sites.2–6,14,15 It is suggested that the dorsal UT ligament should be considered as a separate ligament, based on its different direction and distal attachment site as compared with those of the ulnar collateral ligament (UCL) and ECU subsheath.

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