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CRISPR Loci

CRISPR Loci: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are a specialized region of DNA that contain short, repeating sequences of DNA interspersed with unique spacer sequences.
These loci play a crucial role in the adaptive immune system of prokaryotes, allowing them to recognize and destroy foreign genetic material.
CRISPR loci are widely utilized in genome editing technolgoies, such as the CRISPR-Cas9 system, for precise gene manipulation and modification.
Researchers can leverage CRISPR loci to enable targeted DNA cleavage, gene insertion, deletion, and modification with high efficiency and spceificity.

Most cited protocols related to «CRISPR Loci»

1
Construction of CRISPR Plasmid Library
pDB97 was constructed through phosphorylation and annealing of oligonucleotides B296/B297, followed by ligation in pLZ12spec (ref. 44 (link)) digested by EcoRI/BamHI. We fully sequenced pLZ12spec and deposited its sequence in genebank (accession: KC112384).
pDB98 was obtained after cloning the CRISPR leader sequence was cloned together with a repeat-spacer-repeat unit into pLZ12spec. This was achieved through amplification of crR6Rc DNA with primers B298/B320 and B299/B321, followed by SOEing PCR of both products and cloning in pLZ12spec with restriction sites BamHI/EcoRI. In this way the spacer sequence in pDB98 was engineered to contain two BsaI restriction sites in opposite directions that allow for the scar-less cloning of new spacers.
pDB99 to pDB108 were constructed by annealing of oligonucleotides B300/B301 (pDB99), B302/B303 (pDB100), B304/B305 (pDB101), B306/B307 (pDB102), B308/B309 (pDB103), B310/B311 (pDB104), B312/B313 (pDB105), B314/B315 (pDB106), B315/B317 (pDB107), B318/B319 (pDB108), followed by ligation in pDB98 cut by BsaI.
The pCas9 plasmid was constructed as follow. Essential CRISPR elements were amplified from Streptococcos pyogenes SF370 genomic DNA with flanking homology arms for Gibson Assembly. The tracrRNA and Cas9 were amplified with oligos HC008 and HC010. The leader and CRISPR sequences were amplified HC011/HC014 and HC015/HC009, so that two BsaI type IIS sites were introduced in between two direct repeats to facilitate easy insertion of spacers.
pCRISPR was constructed by subcloning the pCas9 CRISPR array in pZE21-MCS1 through amplification with oligos B298+B299 and restriction with EcoRI and BamHI. The rpsL targeting spacer was cloned by annealing of oligos B352+B353 and cloning in the BsaI cut pCRISPR giving pCRISPR::rpsL.
Jiang W., Bikard D., Cox D., Zhang F, & Marraffini L.A. (2013). CRISPR-assisted editing of bacterial genomes. Nature biotechnology, 31(3), 233-239.
Publication 2013
2',5'-oligoadenylate Arm, Upper Cicatrix Clustered Regularly Interspaced Short Palindromic Repeats CRISPR Loci crRNA, Transactivating Deoxyribonuclease EcoRI Direct Repeat Genome Ligation Oligonucleotide Primers Oligonucleotides Phosphorylation Plasmids
2
CRISPR Array Prediction Limitations
CRISPR arrays were used from published studies or CRISPRdb. They were also predicted with CRISPRFinder, PILER-CR or CRT using the default parameters. The current tools for prediction have some limitations, notably, the lack of prediction of the transcribed strand, the imprecise definition of the DR/Spacer junctions or splitting into several sub arrays.
Biswas A., Gagnon J.N., Brouns S.J., Fineran P.C, & Brown C.M. (2013). CRISPRTarget: Bioinformatic prediction and analysis of crRNA targets. RNA Biology, 10(5), 817-827.
Publication 2013
CRISPR Loci
3
Evaluating CRISPR Spacer Detection Accuracy
Eight genomes containing between zero and seven CRISPR loci were chosen at random from CRISPRdb (20 (link)) (Table 1). Synthetic Illumina data sets (101 bp reads) representing ∼20× coverage were generated for each genome using Grinder 0.4.5 (21 (link)); command-line options: –cf 20 –rd 101 –md poly4. Crass 0.3.1 was run on each data set using a kmer length of 9 (all other parameters default). The spacers identified by Crass were mapped onto the reference genome using blastn 2.2.25+ (22 (link)) to determine whether they were correctly positioned. The spacer graphs for each data set were also analysed to determine whether the ordering of spacers generated by Crass accurately reflected the CRISPR loci found in the original genome assembly.

Specificity and sensitivity analysis of Crass on synthetic short read data sets

Total spacersDetected spacersMissing edgesErroneous edgesSpecificitySensitivity
Bacteroides fragilis YCH46
    CRISPR1973010.63
Acinetobacter sp. ADP1
    CRISPR1660010.83
    CRISPR221210011.00
    CRISPR390882010.98
Sulfolobus solfataricus P2
    CRISPR11021020510.95
    CRISPR294940010.96
    CRISPR331310011.00
    CRISPR495950310.97
    CRISPR5651010.80
    CRISPR622220011.00
    CRISPR765641010.98
Natrialba magadii
    CRISPR127181100.890.58
Helicobacter pylori B800N/AN/A1N/A
Magnetospirillum magneticum AMB-100N/AN/A1N/A
Tsukamurella paurometabola00N/AN/A1N/A
Oligotropha carboxidovorans OM500N/AN/A1N/A
Overall5685531880.990.89

Crass was used to examine synthetic data sets constructed from four genomes that contained between one and seven CRISPR loci, in addition to four genomes that did not contain CRISPRs. The specificity of Crass was calculated by determining the number of detected spacers that did not originate from CRISPRs; the sensitivity was determined by comparing the reconstructed spacer ordering to the ordering found in the genome.

Skennerton C.T., Imelfort M, & Tyson G.W. (2013). Crass: identification and reconstruction of CRISPR from unassembled metagenomic data. Nucleic Acids Research, 41(10), e105.
Publication 2013
CF101 Clustered Regularly Interspaced Short Palindromic Repeats CRISPR Loci Genome Hypersensitivity Pylorus
4
Comprehensive CRISPR Array and Cas Protein Identification
We first used MetaCRT [33 (link)], which we modified from CRT [34 (link)] (to allow detection of partial repeats at the ends of CRISPR arrays), to predict the CRISPR arrays in complete bacterial and archaeal genomes. The genomes were downloaded in October 2016 from the NCBI ftp website (ftp://ftp.ncbi.nlm.nih.gov/genomes/refseq). We focused on complete reference genomes in this study, as CRISPR–Cas systems may be found in separate contigs when draft genomes are used. However, for a few species we analyzed in detail, we augmented the list of genomes with draft genomes: including 13 draft genomes for Streptococcus thermophilus and 4055 draft genomes for Staphylococcus aureus. In some cases, a long CRISPR may be split into multiple ones because of repeats containing excessive mutations or long spacers. To avoid such cases, CRISPRs that are close to each other (<=200 bps) and share very similar repeat sequences were considered to be in the same locus. We then collected the consensus repeat for each putative CRISPR array. We clustered these consensus repeats at 90% sequence identity using CD-HIT-EST [35 (link)]. In this way, a “cluster” contains more than two CRISPR arrays, and a “singleton” refers to the repeats exclusively found within their corresponding CRISPR array.
We then used hmmscan [36 (link)] to search putative proteins found in the genomes against a collection of Cas families to predict putative Cas proteins (using the gathering cutoff). In total, the collection contains 403 Cas families, among which eight were identified from the human microbiomes (using a combination of context-based and similarity-search approaches) [37 ], and 395 were from a recent study [14 (link)]. Since Koonin and colleagues did not build models for the Cas families they curated [14 (link)], we used hmmbuild to construct hmm models for all of their families. Considering that gene prediction is far from perfect for many genomes, for the genomes/contigs that contain CRISPRs but lack cas genes, we further used the FragGeneScan [38 (link)], a gene predictor we have developed for predicting complete as well as fragmented genes in genomic sequences, to re-predict the genes, and then performed cas gene prediction to rule out the possibility of missing cas genes because the genes were not predicted in the first place.
A cas locus defined in this study should contain at least three cas genes, at least one of which belongs to the universal cas genes for CRISPR adaptation (cas1 and cas2) or the main components of interference module including cas7, cas5, cas8, cas10, csf1, cas9, cpf1 [14 (link)].
Zhang Q, & Ye Y. (2017). Not all predicted CRISPR–Cas systems are equal: isolated cas genes and classes of CRISPR like elements. BMC Bioinformatics, 18, 92.
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Publication 2017
Acclimatization Bacteria Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-Associated Protein 9 CRISPR-Cas Systems CRISPR Loci CSF1 protein, human Genes Genome Genome, Archaeal Human Microbiome Mutation Proteins Staphylococcus aureus Streptococcus thermophilus
5
CRISPR Plasmid Construction and Characterization
Supplementary Table S2 contains a list of the plasmids used in this study. Plasmid pDB98 (23 (link)) was used as the source for crRNA guides in S. pneumoniae DB17. This plasmid is a derivative of pLZ12spec (33 (link)) that carries a minimal CRISPR array from S. pyogenes SF370 CRISPR02 containing the leader sequence and two repeats separated by a spacer carrying two BsaI restriction sites for the easy cloning of new spacers. Spacers were cloned by digestion with BsaI, and ligation of annealed oligonucleotides designed as follow: 5′-aaac+(target sequence)+g-3′ and 5′-aaaac+(reverse complement of the target sequence)-3′, where the target sequence is 30 nt and is followed by a functional PAM (NGG). A list of all spacers tested in this study is provided in Supplementary Table S3.
Plasmid pCas9 was generated in a previous study (23 (link)). Plasmid pdCas9 was constructed by introducing D10A and H840A mutations to cas9 on pCas9 through amplification of this plasmid with primers B337/B340 and B338/B339, followed by Gibson assembly (34 (link)) of the two products. Both plasmids contain a minimal CRISPR array with BsaI sites for the cloning of new spacers using complementary oligonucleotides. The in-frame deletion of dcas9 on pdCas9 was achieved by amplification of the plasmid with primers B544/B545 and Gibson assembly of the resulting products.
Fusion of the ω subunit (rpoZ) to dCas9 was achieved by amplification of pdCas9 with primers B441/W551 or B446/W552, and amplification of rpoZ with primers B442/W550 or W553/B448 to create the C- or N-terminal ω fusions, respectively, followed by Gibson assembly. Plasmid pWJ66 carries the C-terminal fusion and pWJ68 the N-terminal fusion. To measure induction in E. coli KS1ΔZ, a chloramphenicol-resistant strain, the plasmid resistance was changed to spectinomycin. Plasmids pWJ66 and pWJ68 were amplified with oligos H001/H002, and the spectinomycin resistance gene was amplified from pSWKspec with oligos H003/H004; Gibson assembly of the PCR products generated plasmids pDB191 and pDB192.
Plasmid pDB127 was constructed by amplification of gfp-mut2 (35 (link)) with primers B368/B371, followed by digestion with EcoRI and BamHI, and ligation together with the annealed oligonucleotides B369/B370 in the pZS24-MCS1 (36 (link)) vector cut with XhoI and BamHI. The sequence of the PAM-rich promoter carried by pDB127 is provided in the Supplementary Sequences.
Plasmids pWJ89, pWJ96 and pWJ97 were constructed by changing the promoter of gfp-mut2 on pDB127 for biobrick promoters BBa_J23117, BBa_J23116 and BBa_J23110, respectively (http://partsregistry.org). The region upstream of the promoter was changed to include multiple NGG PAM sequences on both strands. Full sequences of these promoters are provided in the Supplementary Sequences.
Bikard D., Jiang W., Samai P., Hochschild A., Zhang F, & Marraffini L.A. (2013). Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Research, 41(15), 7429-7437.
Publication 2013
2',5'-oligoadenylate Chloramphenicol Cloning Vectors CRISPR Loci Deletion Mutation Deoxyribonuclease EcoRI Digestion Escherichia coli Genes Ligation Mutation Oligonucleotide Primers Oligonucleotides Plasmids Protein Subunits Pulmonal S Reading Frames RNA, CRISPR Guide Signal Peptides Spectinomycin Strains Streptococcus pyogenes

Most recents protocols related to «CRISPR Loci»

1
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].
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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
2
Exploring Phage Defense Systems

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Publication 2023
Bacteria Bacteriophages CRISPR Loci Phenotype Prophages Strains Superinfection Technique, Dilution Virulence
3
Genomic Analysis of CRISPR Systems
Analysis of CRISPR‐positive complete genomes and high‐quality assemblies was performed to better characterize the genomic context surrounding the cas gene sets and/or CRISPR arrays. High‐quality assemblies with at least 4 kb flanking the cas gene sets were considered. These regions were annotated by Prokka (https://github.com/tseemann/prokka) (Seemann, 2014 (link)). Synteny was established by either the Mauve algorithm (http://darlinglab.org/mauve/mauve.html) (Darling et al., 2010 (link)) or visual inspection of annotated proteins.
Scrascia M., Roberto R., D'Addabbo P., Ahmed Y., Porcelli F., Oliva M., Calia C., Marzella A, & Pazzani C. (2023). Bioinformatic survey of CRISPR loci across 15 Serratia species. MicrobiologyOpen, 12(2), e1339.
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Publication 2023
Clustered Regularly Interspaced Short Palindromic Repeats CRISPR Loci Genes Genome Proteins Synteny
4
Comparative Genomics of Serratia Bacteria
One hundred and forty‐six Serratia complete genomes were considered in this study. The set of genomes encompasses the 15 S. marcescens complete genomes we previously analyzed (Scrascia et al., 2019 (link)) and those of the genus Serratia available at the CRISPR–Cas++ database (https://crisprcas.i2bc.paris-saclay.fr/MainDb/StrainList) up to December 12, 2020 (Couvin et al., 2018 (link); Pourcel et al., 2020 (link)) (Supporting Information: Table S1). Among genome sequences available at the assembly level of scaffolds or contigs available at the National Center for Biotechnology Information database (NCBI) (https://www.ncbi.nlm.nih.gov/assembly) up to December 12, 2020, we selected the high‐quality assemblies (N50 > 50 kb, i.e. 50% of the entire assembly is contained in contigs or scaffolds equal to or larger than the 50 kb) that have been included in the study.
Species attribution and strain details (name, place, date of isolation) were recovered (when available) from GenBank or related articles. Serratia strains AS12 (NC_015566.1), FGI94 (NC_020064), FS14 (NZ_CP005927), SCBI (NZ_CP003424), YD25 (NZ_CP016948), and DSM21420 (GCA_000738675) were reclassified as reported by Sandner‐Miranda et al. (2018 (link)), Sandner‐Miranda et al. (2018 (link)). In the study reported by Sandner‐Miranda et al., the strain ATCC39006 was not assigned to the genus Serratia and we did not include it in this study.
We also included sequences with the accessions MK507743, MK507744, MK507745, and MK507746 referring to contigs (N50 ranging from 228817 to 291462) harboring CRISPR loci in genome assemblies (unpublished) of four S. marcescens strains reported as secondary symbionts in the Red Palm Weevil (RPW) Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae) (Scrascia et al., 2016 (link), 2019 (link)) (Supporting Information: Table S1), an alien invasive pest now threatening South America (Dalbon et al., 2021 (link)).
Scrascia M., Roberto R., D'Addabbo P., Ahmed Y., Porcelli F., Oliva M., Calia C., Marzella A, & Pazzani C. (2023). Bioinformatic survey of CRISPR loci across 15 Serratia species. MicrobiologyOpen, 12(2), e1339.
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Publication 2023
Aliens Arecaceae Beetles Clustered Regularly Interspaced Short Palindromic Repeats CRISPR Loci Genome isolation Plague Serratia Strains Weevils
5
Comprehensive CRISPR-Cas System Characterization
Details about the detection of a cas gene cluster with associated arrays (CRISPR–Cas system) and CRISPR arrays only for complete genomes were retrieved from the CRISPR–Cas++ database. CRISPR arrays recorded by CRISPR–Cas++ were assigned to Levels 1–4 based on the criteria required to select the minimal structure of putative CRISPR as reported by Pourcel et al. (2020 (link)). Level 1 is the lowest level of confidence. Levels 2–4 were assigned based on the conservation of repeats (which must be high in a real CRISPR) and on the similarity of spacers (it must be low). Level 4 CRISPRs were defined as the most reliable ones. Levels 1–3 may correspond to false CRISPRs. In our study, only CRISPRs recorded with Level 4, were considered. CRISPRs without a set of cas genes in the host genome were defined as “orphans.” Genomes harboring cas gene clusters were then submitted to the CRISPRone analysis suite (http://omics.informatics.indiana.edu/CRISPRone/) (Zhang & Ye, 2017 (link)) to graphically visualize the architecture of each cluster. The same suite was used to search and visualize cas gene clusters in the high‐quality assemblies. A subtype of cas gene clusters was assigned according to the recent classification update for CRISPR–Cas systems (Makarova et al., 2020 (link)).
Scrascia M., Roberto R., D'Addabbo P., Ahmed Y., Porcelli F., Oliva M., Calia C., Marzella A, & Pazzani C. (2023). Bioinformatic survey of CRISPR loci across 15 Serratia species. MicrobiologyOpen, 12(2), e1339.
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Publication 2023
Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-Cas Systems CRISPR Loci Gene Clusters Genes Genome Orphaned Children

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More about "CRISPR Loci"

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a revolutionary genome editing technology that has transformed the field of molecular biology.
These specialized regions of DNA contain short, repeating sequences interspersed with unique spacer sequences, playing a crucial role in the adaptive immune system of prokaryotes.
CRISPR loci, the specific DNA sequences that make up the CRISPR system, are widely utilized in cutting-edge genome editing technologies, such as the CRISPR-Cas9 system.
This powerful tool enables precise gene manipulation, allowing for targeted DNA cleavage, gene insertion, deletion, and modification with unparalleled efficiency and specificity.
Researchers leveraging CRISPR loci can harness the power of this technology to conduct a wide range of experiments, from gene expression studies to disease modeling and therapeutic development.
Techniques like Lipofectamine 3000 and Phusion DNA polymerase are often employed to facilitate the delivery and amplification of CRISPR components, while MiSeq sequencing and QIAquick PCR Purification Kits can be used to analyze the outcomes of CRISPR-mediated modifications.
Furthermore, the DNeasy Blood & Tissue Kit and TrueSeq Nano DNA Library Prep protocol can be utilized to extract and prepare high-quality DNA samples for CRISPR-based experiments.
The QIAquick Gel Extraction Kit and BD Influx cell sorter can also play a crucial role in the purification and isolation of CRISPR-modified cells or DNA fragments.
To optimize CRISPR loci research, scientists can leverage AI-driven platforms like PubCompare.ai.
This innovative tool helps researchers identify the best protocols and products from the literature, preprints, and patents, boosting reproducibility and accuracy in their CRISPR experiments.
By harnessing the power of CRISPR loci and the latest technological advancements, researchers can push the boundaries of genetic engineering and unlock new frontiers in the life sciences.
One typo: 'technolgoies' instead of 'technologies'.