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Clustered Regularly Interspaced Short Palindromic Repeats

Clustered Reglularly Interspaced Short Palindromic Repeats (CRISPR) are a family of DNA sequences found in the genomes of prokaryotic organisms, such as bacteria and archaea.
These repeats play a crucial role in the adaptive immune system, allowing these organisms to detect and destroy the DNA of invading viruses.
The CRISPR sequences are interspersed with short unique sequences called 'spacers,' which are derived from foreign genetic material.
When a virus infects a cell, the CRISPR system recognizes the viral DNA and uses it to create small CRISPR RNA (crRNA) molecules that guide Cas (CRISPR-associated) proteins to the viral DNA, leading to its destruction.
This revolutionary system has been widely adopted as a powerful genome editing tool, enabling researchers to precisely modify genes in a variety of organisms, including human cells, with unparalleled efficiency and precision.

Most cited protocols related to «Clustered Regularly Interspaced Short Palindromic Repeats»

1
Automated CRISPR Identification Pipeline
CRISPRFinder core routines were developed in Perl under Debian Linux. The input of the web tool is a genomic query sequence of length up to 67 Mb in ‘FASTA’ format. Possible locations of CRISPRs (consisting of at least one motif) are detected by finding maximal repeats. A maximal repeat (26 ) is a repeat that cannot be extended in either direction without incurring a mismatch. The total number of maximal repeats in a sequence of size n is linear (less than n) which is interesting since the computation may be done in linear time using a suffix-tree-based algorithm. A CRISPR pattern of two DRs and a spacer may be considered as a maximal repeat where the repeated sequences are separated by a sequence of approximately the same length.
The operation of the program can be divided into four main steps summarized in Figure 1: (Step 1) browsing the maximal repeats of length 23–55 bp interspaced by sequences of 25–60 bp, (Step 2) selecting the DR consensus according to a defined score taking into account the number of occurrences of the candidate DR in the whole genome and privileging internal mismatches between the DRs rather than mismatches in the first or the last nucleotides, (Step 3) defining candidate CRISPRs after checking if they fit CRISPR definition, (Step 4) eliminating residual tandem repeats.

CRISPR Finder flow chart. (Step 1) Browsing the maximal repeats to get possible CRISPR localizations using the Vmatch program. (Step 2) Consensus DR selection according to candidate occurrences and a score computation: the score privileges internal mismatches between direct repeats of a cluster rather than boundary mismatches. (Step 3) DR and spacers size check. (Step 4) Tandem repeats elimination using ClustalW for aligning spacers.

In the first step, maximal repeats are found by the software Vmatch (http://www.vmatch.de/), the upgrade of REPuter (22–24 ). Vmatch is based on a comprehensive implementation of enhanced suffix arrays (27 ) which provides the power of suffix trees with lower space requirements. A one nucleotide mismatch is allowed permitting minimal CRISPRs with a single nucleotide mutation between DRs to be found. Hereafter, the obtained maximal repeats are grouped to define regions of possible CRISPRs with a display of consensus DR candidates related to each cluster.
The second step is aimed at retrieving the DR consensus of each cluster. The difficulty resides especially in the identification of boundaries, which is very important to extract the correct spacers and compare DRs. In fact, the consensus DR is selected as the maximal repeat which occurs the most in the whole underlying genome sequence with respect to the forward and the reverse complement directions (since two CRISPRs having the same DR consensus may be in opposite directions). Thus, ambiguity in the choice of a DR will be eliminated in the case of presence of similar DRs in other CRISPRs of the related genomic sequence. However, if occurrence numbers are equal, more than a single DR consensus candidate are kept and later compared. Given a candidate consensus DR, the pattern search program fuzznuc of the EMBOSS package (28 (link)) is applied to get DRs’ positions in the related cluster. As the first or the last DR in a CRISPR may be diverged/truncated, a mismatch of one-third of the DR length is allowed between the flanking DRs and the candidate consensus DR, whereas smaller nucleotide differences are allowed between the other DRs to take into account possible single mutations. In case of multiple DR candidates, a score is computed and the best one (minimum) is picked. This score favours candidates which are encountered more frequently, rather than consensus DR showing less internal mismatches.
Once the DR consensus is determined, the corresponding spacers (Step 3) are extracted according to the DR boundaries determined previously. The spacer length is not allowed to be shorter than 0.6 or longer than 2.5 times the DR length. These sizes are in the range of CRISPRs described in the literature.
The last step consists in discarding false CRISPRs. Therefore, tandem repeats are eliminated by comparing the consensus DR with the spacer if there is only one spacer, or by comparing spacers between each other. The comparison is done with the CLUSTALW program (29 (link)) and the percentage of identity between spacers is not allowed to exceed 60%. Finally, candidates having at least three motifs and at least two exactly identical DRs are considered as confirmed CRISPRs. The remaining candidates are considered as questionable. These should be critically investigated by, for example, checking for intraspecies size variation of the locus.
Grissa I., Vergnaud G, & Pourcel C. (2007). CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Research, 35(Web Server issue), W52-W57.
Publication 2007
Clustered Regularly Interspaced Short Palindromic Repeats Direct Repeat Genome Mutation Nucleotides Repetitive Region Tandem Repeat Sequences Trees
2
Identifying Essential Genes in CRISPR Screens
RIGER was originally designed to identify essential genes in genome-scale shRNA screens using microarray technology [14 (link)]. To accommodate the input requirements of RIGER, we median-normalized (the same as the first step of MAGeCK) and log2 transformed read counts from CRISPR/Cas9 knockout screens. We ran the latest version of RIGER (0.1 beta) as specified in the paper [14 (link)] and website [37 ]. Default RIGER parameters were used in all experiments, except that we set the number of permutations to 100,000 to get a more precise P-value. The results were ranked by the P-values of the genes.
Li W., Xu H., Xiao T., Cong L., Love M.I., Zhang F., Irizarry R.A., Liu J.S., Brown M, & Liu X.S. (2014). MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biology, 15(12), 554.
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Publication 2014
Clustered Regularly Interspaced Short Palindromic Repeats Genes Genes, Essential Genome Microarray Analysis Short Hairpin RNA
3
CRISPR-Cas9 Genome Editing Profiling
HEK 293FT cells plated in 96-well plates were transfected with Cas9 plasmid DNA and sgRNA PCR cassette 72 h before genomic DNA extraction (Supplementary Fig. 4). The genomic region flanking the CRISPR target site for each gene was amplified (Supplementary Fig. 6, Supplementary Table 5 and Supplementary Sequences) by a fusion PCR method to attach the Illumina P5 adapters as well as unique sample-specific barcodes to the target amplicons (schematic described in Supplementary Fig. 5). PCR products were purified using EconoSpin 96-well Filter Plates (Epoch Life Sciences) following the manufacturer's recommended protocol.
Barcoded and purified DNA samples were quantified by Quant-iT PicoGreen dsDNA Assay Kit or Qubit 2.0 Fluorometer (Life Technologies) and pooled in an equimolar ratio. Sequencing libraries were then sequenced with the Illumina MiSeq Personal Sequencer (Life Technologies).
Hsu P.D., Scott D.A., Weinstein J.A., Ran F.A., Konermann S., Agarwala V., Li Y., Fine E.J., Wu X., Shalem O., Cradick T.J., Marraffini L.A., Bao G, & Zhang F. (2013). DNA targeting specificity of RNA-guided Cas9 nucleases. Nature biotechnology, 31(9), 827-832.
Publication 2013
Biological Assay Cells Clustered Regularly Interspaced Short Palindromic Repeats DNA, Double-Stranded EPOCH protocol Genes Genome H-DNA PicoGreen Plasmids
4
CRISPR-Cas9 Genome Editing in Cells
For human cells, expression vector PX330 (Addgene plasmid 42230) encoding Cas9 and chimeric guide RNA was used (11 (link)). The LBR guides were cloned into expression vector pBluescript with the sgRNA cassette of PX330 and transfected into the K562 line stably transformed with Cas9. For Drosophila cells, Cas9 expression vector pBS-Hsp70-Cas9 (Addgene plasmid 46294) was used in combination with pU6-BbsI-chiRNA construct (Addgene plasmid 45946) (12 (link)). The sgRNAs were designed using CRISPR design (http://crispr.mit.edu/) (13 (link)) and CHOPCHOP (https://chopchop.rc.fas.harvard.edu/) (14 (link)).
The following sgRNA sequences were used:
For the cloning of individual DNA fragments from the edited GFP gene, PCR products were ligated in Zero Blunt vector (Invitrogen) using standard procedures.
Brinkman E.K., Chen T., Amendola M, & van Steensel B. (2014). Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Research, 42(22), e168.
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Publication 2014
Cells Chimera Cloning Vectors Clustered Regularly Interspaced Short Palindromic Repeats Drosophila Genes Heat-Shock Proteins 70 Homo sapiens Plasmids
5
CRISPOR: Comprehensive CRISPR Target Scoring
CRISPOR uses the popular BWA aligner [35 (link)] version 0.7.5a-r405 in iterative mode (“-N”). All genomic hits within a certain edit distance are retrieved from BWA, filtered for the requested PAM sequence, and scored and annotated with gene model information using the UCSC Genome Browser command line tools [48 (link)]. CRISPOR ignores off-targets with an off-target score <0.1 for the PAM NGG and those with a score <1.0 for the PAMs NAG and NGA (Fig. 2). Based on all off-target scores for a guide, a specificity score is calculated using the same formula as on the CRISPR Design website (http://crispr.mit.edu).
CRISPOR currently supports 113 genomes. Potential off-targets can be filtered to retain only those in exons, those that may be of concern when isolating cell clones, or those located on the same chromosome as the target, whose mutations may co-segregate and, therefore, confound phenotypic analysis when studying genetically modified organisms. The predicted guides and their off-targets are shown as a table, with links to the Ensembl and UCSC genome browsers. Results can be downloaded as spreadsheet files for archiving. Several features of practical interest are included, such as primer sequences for cloning into Addgene plasmids, direct expression with T7 RNA polymerase, or PCR amplification of the genome sequence targeted for T7 assays.
All scoring functions have been devised for S. pyogenes Cas9 only. Pending further experimental investigation, we have applied scores to engineered S. pyogenes Cas9 as well as to other Cas9 proteins shown to work in mammalian cells. The VQR Cas9 mutant was chosen because it discriminates best between NGA and NGG PAMs. The CRISPOR specificity score, similar to the situation with wild-type Cas9, was therefore calculated by ignoring off-targets with an off-target score <0.1 for the PAM NAG and those with a score <1.0 for the PAMs NGG and NGA.
Haeussler M., Schönig K., Eckert H., Eschstruth A., Mianné J., Renaud J.B., Schneider-Maunoury S., Shkumatava A., Teboul L., Kent J., Joly J.S, & Concordet J.P. (2016). Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biology, 17, 148.
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Publication 2016
Biological Assay Cells Chromosomes Clustered Regularly Interspaced Short Palindromic Repeats Exons F11R protein, human GDF15 protein, human Genome Mammals Mutation Oligonucleotide Primers Organisms, Genetically Modified Phenotype Plasmids Proteins Streptococcus pyogenes Transcription, Genetic

Most recents protocols related to «Clustered Regularly Interspaced Short Palindromic Repeats»

1
CRISPR-Cas9 RNA Editing in iPSCs
Not available on PMC !

Example 2

iPS cells were prepared according to protocols known in the art and seeded in a Geltrex®-Matrix coated 12-well culture dish. Transfection was performed in iPSCs with 3 ul of Lipofectamine® 2000 or 3 ul of Lipofectamine® 3000 as indicated and according to manufacturer's instructions, to deliver a GeneArt® CRISPR Nuclease vector targeting the HPRT locus. Transfection was also performed with GeneArt® CRISPR Nuclease RNA editing system targeting the HPRT locus and 3 ul of Formulation 21 lipid aggregate complex. RNA editing system utilizes a Cas9 mRNA, which was prepared via in vitro transcription with the Ambion® mMESSAGE mMACHINE® Kit, and a gRNA which was transcribed using the Ambion® MEGAshortscript™ Kit. Cells were harvested 72-hours post-transfection and cleavage efficiency was determined using the GeneArt® Genomic Cleavage Detection Kit.

Results are shown in FIG. 2A and FIG. 2B, which clearly demonstrate that using an mRNA based form of Cas9 with a guide-RNA for gene editing with the lipid aggregates described herein for transfection results in at least 4-fold more targeted cleavage of the host cell genome when compared to standard DNA based editing approaches.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

US11872285B2. Compositions and methods for efficient delivery of molecules to cells (2024-01-16). LIFE TECHNOLOGIES CORPORATION [US]. Inventors: Xavier de Mollerat du Jeu [US], Nektaria Andronikou [US].
Full text: Click here
Patent 2024
Cells Cloning Vectors Clustered Regularly Interspaced Short Palindromic Repeats Cytokinesis Genes Genome Hyperostosis, Diffuse Idiopathic Skeletal Induced Pluripotent Stem Cells Lipids Lipofectamine lipofectamine 2000 RNA, Messenger Transcription, Genetic Transfection
2
Interferon Signaling in ADAR1-Dependent Cancer Cells
Not available on PMC !

Example 9

An analysis of gene ontology (GO) categories associated with ADAR1 dependent cells revealed that NCI-H1650 and HCC366 (“HCC-366”), two ADAR1 dependent cell lines, both have elevated basal expression of interferon inducible genes (FIG. 35). The expression levels of interferon-inducible genes were also elevated in NCI-H196 cells (FIG. 36).

In light of the correlation between ADAR1 dependency and the expression of interferon-inducible genes, additional cancer cell lines from the Molecular Signatures Database (MSigDB) (Liberzon et al. (2015) Cell Systems 1:417-425) was examined. Cancer Cell Line Encyclopedia (CCLE) clustering was performed based on the Type I/Interferon-a gene set, which contained 97 genes including PKR. The resulting cluster included HCC366, NCI-H1650 and 9 additional lung cell lines. Among these cell lines, HCC1438 and NCI-H596 were sensitive to knockout of ADAR1 by lentiviral CRISPR-Cas9 (FIG. 37).

All the above-identified ADAR1 dependent cancer cell lines showed elevated interferon signaling markers, e.g., phosphorylation of STAT1 and expression of interferon-stimulated gene (ISGs) (FIG. 38). Elevated interferon signaling in the ADAR1 dependent cancer cell lines did not necessarily lead to PD-L1 overexpression (FIG. 38). Cell lines in the high interferon signaling cluster (LN215_CENTRAL_NERVOUS_SYSTEM, NCIH596_LUNG, HCC1438_LUNG, T3M10_LUNG, NCIH1869_LUNG, SW900_LUNG, HCC366_LUNG, SKLU1_LUNG, NCIH1650_LUNG, HCC4006_LUNG, and NCIH1648_LUNG) displayed high IFN-β, but not IFN-α (FIG. 39). As such, cancer cell lines sensitive to ADAR1 or ISG15 knockdown displayed elevated interferon secretion and downstream signaling. To further investigate the relationship between ADAR1 and IFN-β secretion, it was found that ADAR1 knockout led to amplified IFN-β secretion in cell lines primed with high basal interferon activation (FIG. 40). It was also found that ADAR1 dependent cell lines do not show enhanced sensitivity to IFN-α or IFN-β alone (FIG. 41).

US11873486B2. Modulating dsRNA editing, sensing, and metabolism to increase tumor immunity and improve the efficacy of cancer immunotherapy and/or modulators of intratumoral interferon (2024-01-16). Dana-Farber Cancer Institute, Inc. [US]. Inventors: Matthew Meyerson [US], William N. Haining [US], Jeffrey Ishizuka [US], Robert Manguso [US], Hugh Gannon [US].
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Patent 2024
CD274 protein, human Cell Lines Cells Central Nervous System Clustered Regularly Interspaced Short Palindromic Repeats Gene Expression Genes Hypersensitivity Interferon-alpha Interferons Interferon Type I Light Lung Malignant Neoplasms Phosphorylation secretion STAT1 protein, human
3
In Vivo Testing of CRISPR Therapies
Not available on PMC !

Example 96

After the CRISPR-Cas9/DNA donor combinations have been re-assessed, the lead formulations will be tested in vivo in an animal model. Suitable animal models include, by way of non-limiting example a FGR mouse model with the livers repopulated with human hepatocytes or iPSC derived hepatocytes (normal or G6PC deficient).

Culture in human cells allows direct testing on the human target and the background human genome, as described above.

Preclinical efficacy and safety evaluations can be observed through engraftment of modified mouse or human hepatocytes in FGR mice. The modified cells can be observed in the months after engraftment.

US11866727B2. Materials and methods for treatment of glycogen storage disease type 1A (2024-01-09). CRISPR THERAPEUTICS AG [None]. Inventors: Chad Albert Cowan [US], Roman Lvovitch Bogorad [US], Ante Sven Lundberg [US], Kirsten Leah Beaudry [US].
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Patent 2024
Animal Model Cell Culture Techniques Cells Clustered Regularly Interspaced Short Palindromic Repeats Genome, Human Hepatocyte Homo sapiens Induced Pluripotent Stem Cells Liver Mus Safety Tissue Donors
4
Optimizing CRISPR-Cas9 for Hepatocyte Editing
Not available on PMC !

Example 95

After testing the different strategies for HDR gene editing, the lead CRISPR-Cas9/DNA donor combinations will be re-assessed in primary human hepatocytes for efficiency of deletion, recombination, and off-target specificity. Cas9 mRNA or RNP will be formulated into lipid nanoparticles for delivery, sgRNAs will be formulated into nanoparticles or delivered as AAV, and donor DNA will be formulated into nanoparticles or delivered as AAV.

US11866727B2. Materials and methods for treatment of glycogen storage disease type 1A (2024-01-09). CRISPR THERAPEUTICS AG [None]. Inventors: Chad Albert Cowan [US], Roman Lvovitch Bogorad [US], Ante Sven Lundberg [US], Kirsten Leah Beaudry [US].
Full text: Click here
Patent 2024
Clustered Regularly Interspaced Short Palindromic Repeats Deletion Mutation Hepatocyte Homo sapiens Lipid Nanoparticles Obstetric Delivery Recombination, Genetic RNA, Messenger Tissue Donors
5
CasX CRISPR Plasmid Interference in E. coli
Not available on PMC !

Example 2

FIGS. 4A-4C. Plasmid Interference by CasX expressed in E. coli. Experimental design of CasX plasmid interference. Competent E. coli cells expressing the minimal interference CasX locus (acquisition proteins removed) were prepared. These cells were transformed with a plasmid containing a match to the spacer in the CasX CRISPR locus (target) or not (non-target) and plated on media containing antibiotic selection for the CRISPR and target plasmid. Successful plasmid interference results in reduced number of transformed colonies for the target plasmid. FIG. 4B cfu/ug of transformed plasmid containing spacer from CasX1 (sX1), spacer from CasX2 (sX2) or a non-target plasmid containing a random 30 nt sequence. FIG. 4C serial dilution was performed of transformants from FIG. 4B on media containing antibiotic selection for both the CRISPR and target plasmid.

FIGS. 5A-5B PAM dependent plasmid interference by CasX. PAM depletion assays were conducted with CasX. E. coli containing the CasX CRISPR locus were transformed with a plasmid library with 7 nucleotides randomized 5′ or 3′ of the target sequence. The target plasmid was selected for and transformants were pooled. The randomized region was amplified and prepared for deep sequencing. Depleted sequences were identified and used to generate a PAM logo. FIG. 5B PAM logo generated for deltaproteobacteria CasX showed a strong preference for sequences containing a 5′-TTCN-3′ flanking sequence 5′ of the target. A 3′ PAM was not detected. c, PAM logo generated for planctomyces CasX showed a strong preference for sequences containing a 5′-TTCN-3′ flanking sequence 5′ of the target with lower stringency at the first T. A 3′ PAM was not detected.

FIGS. 6A-6C. CasX is a dual-guided CRISPR-Cas effector complex. FIG. 6A CRISPR locus for tracrRNA knockout experiments and sgRNA tests. FIG. 6B colony forming units (cfu) per g of transformed plasmid containing a target or non-target sequence. Deletion of the tracrRNA resulted in ablation of plasmid interference. Expression of a synthetic sgRNA in place of the tracrRNA and CRISPR array resulted in robust plasmid interference by CasX. FIG. 6C diagram of sgRNA design (derived from tracrRNA and crRNA sequences for CasX1). The tracrRNA (green) was joined to the crRNA (repeat, black; spacer, red) by a tetraloop (GAAA).

FIG. 7. Schematic of CasX RNA guided DNA interference. CasX binds to a tracrRNA (green) and the crRNA (black, repeat; red, spacer). Base pairing of the guide RNA to the target sequence (blue) containing the correct protospacer adjacent motif (yellow) results in double stranded cleavage of the target DNA. The depicted sequences are derived from tracrRNA and crRNA sequences for CasX1.

US11873504B2. RNA-guided nucleic acid modifying enzymes and methods of use thereof (2024-01-16). The Regents of the University of California [US]. Inventors: Jennifer A Doudna [US], Jillian F. Banfield [US], David Burstein [US], Lucas Benjamin Harrington [US], Steven C. Strutt [US].
Full text: Click here
Patent 2024
Antibiotics Biological Assay Cells Clustered Regularly Interspaced Short Palindromic Repeats CRISPR Loci crRNA, Transactivating Deletion Mutation Deltaproteobacteria DNA Cleavage DNA Library Enzymes Escherichia coli Nucleic Acids Nucleotides Plasmids Proteins RNA, CRISPR Guide Technique, Dilution

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More about "Clustered Regularly Interspaced Short Palindromic Repeats"

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are a revolutionary genome editing technology that have transformed the field of molecular biology.
These DNA sequences, found in the genomes of prokaryotic organisms like bacteria and archaea, play a crucial role in their adaptive immune system.
The CRISPR system works by using short, unique sequences called 'spacers' that are derived from foreign genetic material, such as viral DNA.
When a virus infects a cell, the CRISPR system recognizes the viral DNA and uses it to create small CRISPR RNA (crRNA) molecules.
These crRNA molecules then guide CRISPR-associated (Cas) proteins to the viral DNA, leading to its destruction.
This powerful system has been widely adopted by researchers as a highly efficient and precise tool for gene editing.
Using CRISPR, scientists can now modify genes in a variety of organisms, including human cells, with unprecedented accuracy.
To facilitate CRISPR research, various tools and reagents have been developed, such as Lipofectamine 2000 and Lipofectamine 3000 for transfection, LentiCRISPR v2 for lentiviral delivery, and Puromycin for selection.
Additionally, the use of cell culture media like DMEM, supplemented with FBS and Penicillin/Streptomycin, has become commonplace in CRISPR experiments.
By harnessing the power of CRISPR and these complementary technologies, researchers are able to push the boundaries of genetic engineering and make groundbreaking discoveries.
Whether you're studying gene function, developing therapeutic interventions, or exploring the fundamental mechanisms of life, CRISPR has become an indispensable tool in the modern biological laboratory.