CRISPRlnc
Non-coding gene CRISPR/Cas9 sgRNA one-stop design tool
1. Overview of CRISPRlnc website
CRISPRlnc is a new machine learning method, for designing lncRNA-specific sgRNA in CRISPR/Cas9 system. CRISPRlnc was trained based on noncoding datasets of two different mechanisms, namely CRISPR knock-out (CRISPRko) and CRISPR inhibition (CRISPRi) to capture the different characterization preferences of on-target validity under these two mechanisms. The output results of CRISPRlnc include the sequence, genome localization, on-target activity score (cutting effectiveness), and off-target risk of each candidate sgRNA. For the CRISPRko mechanism, we recommend users to select paired sgRNAs located in the gene body region with effective cleavage activity and lower off-target risk. For the CRISPRi mechanism, we recommend users to select single sgRNAs located in the promoter region with effective cleavage activity and low off-target risk.
The overview of CRISPRlnc
2. CRISPR knock-out Design(paired-sgRNA)
In the design of sgRNA based on CRISPRko, we added the function of paired sgRNA design, as the CRISPRko mechanism for lncRNA prefers to knock out large fragments of gene body region through paired sgRNAs working together. As shown on the below figure, follow the steps to select the CRISPR mechanism and reference genome (we provide nine reference genomes including human, mouse, and zebrafish), then enter the sequence of the gene for which the CRISPR sgRNA is to be designed, select the desired promoter length, and click Submit. We support entering multiple sequences at the same time. In addition to the gene sequence, you can also choose to input the ID or symbol of the gene.
CRISPRko input
Step 1 - Select the reference genome by clicking.We provide nine reference genomes: human, mouse, Drosophila melanogaster, zebrafish, paniscus, gorilla, monkey, cow, and goat.
Step 2 - Select the CRISPR knock-out mechanism.
Step 3 - Select the length of the gene fragments to be deleted.The simultaneous action of two sgRNA-guided Cas9 enzymes produces large base deletions that are more likely to knock out non-coding genes, and this option allows the user to define the distance that two sgRNAs are spaced apart on the genome.
Step 4 - Select the length of the promoter region by pulling the progress bar. Instead of having to manually enter a promoter sequence, we will automatically return a promoter sequence of the appropriate length based on the gene entered by the user. Maximum length is 2000.
Step 5 - Select the length of the downstream region of the gene by pulling the progress bar.
Step 6 - Choose between sequence or ID input.we support ensemble ID and gene symbol.
Step 7 - Paste sequence or paste ID.
Step 8 - Select whether off-target results are required.Genome-wide off-target calculations take more time, and we support sgRNA designs that only calculate on-target activity.
Step 9 - Select whether or not you want to filter poorly performing targets.Some targets with too high off-target risk or poor on-target activity can be filtered out directly and not displayed on the results page..
Step 10 - Click submit.
Results:
CRISPRko results
Input information:Shows information about the input gene, making it easy to view multiple transcripts of the gene (if they exist).
sgRNA overview:Shows the distribution of all targets on the sequence.
sgRNA details:
Column 1: transcript.Users can click to change to another target transcript.
Column 2: distance Distance between two sgRNA spacers
Column 3: gene region The relative location of the sgRNA to the target gene. Targets located in the gene body region are generally considered more suitable for knockdown.
Column 4: ID sgRNA ID.
Column 5: start The position of this sgRNA relative to the gene start site.' +' indicates after the transcription start site and' - 'indicates before the transcription start site.
Column 6: sequence target site sequence.
Column 7: PAM target site PAM sequence.
Column 8: GC sequence GC content (%) of the target site.
Column 9: position The location of the target on the chromosome. The user can view it on the Genome Browser by clicking on it.
Column 10: genomic location The location of the gene at which the target is located.
Column 11: biotype Identifies whether the target is located in an intron or exon, or in an intergenic region.
Column 12: strand the strand of target sequence in the giving sequence. ("-" or "+")
Column 13: cutting effectiveness Identifies the on-target activity of the target, which is calculated by the SVM algorithm, and the higher the activity index, the easier the target is to cut.
Column 14: off-target risk Identifies the potential off-target risk of the target, the higher it is the more likely the target will be off-target and is not recommended.
Column 15: off-target detailDetailed off-target information for the target can be viewed by clicking on it.
Column 16: pair score Overall score of the target pair. The target area, on-target activity and off-target risk are taken into account, with higher scores indicating a higher recommendation.
3. CRISPR inhibition Design
When designing sgRNA for CRISPRi, we will generate the promoter sequence of the lncRNA gene based on the user input gene and promoter length. This will allow us to design sgRNA that targets the promoter region of the lncRNA gene, which inhibits its transcription. It is important to note that the CRISPRi mechanism has a tendency to target the promoter region of the lncRNA gene. As with the knock-out mechanism, user can choose to enter a gene ID or a gene sequence.
CRISPRi input
Step 1 - Select the reference genome by clicking.We provide nine reference genomes: human, mouse, Drosophila melanogaster, zebrafish, paniscus, gorilla, monkey, cow, and goat.
Step 2 - Select the CRISPR inhibition mechanism.
Step 3 - Select the length of the promoter region by pulling the progress bar. Instead of having to manually enter a promoter sequence, we will automatically return a promoter sequence of the appropriate length based on the gene entered by the user. Maximum length is 2000.
Step 4 - Select the length of the downstream region of the gene by pulling the progress bar.
Step 5 - Choose between sequence or ID input.we support ensemble ID and gene symbol.
Step 6 - Paste sequence or paste ID.
Step 7 - Select whether off-target results are required.Genome-wide off-target calculations take more time, and we support sgRNA designs that only calculate on-target activity.
Step 8 - Select whether or not you want to filter poorly performing targets.Some targets with too high off-target risk or poor on-target activity can be filtered out directly and not displayed on the results page..
Step 9 - Click submit.
Results:
CRISPRi results
Input information:Shows information about the input gene, making it easy to view multiple transcripts of the gene (if they exist).
sgRNA overview:Shows the distribution of all targets on the sequence.
sgRNA details:
Column 1: transcript.Users can click to change to another target transcript.
Column 2: gene region The relative location of the sgRNA to the target gene. Targets located in the promoter region are generally considered more suitable for gene inhibition.
Column 3: ID sgRNA ID.
Column 4: start The position of this sgRNA relative to the gene start site.' +' indicates after the transcription start site and' - 'indicates before the transcription start site.
Column 5: sequence target site sequence.
Column 6: PAM target site PAM sequence.
Column 7: GC sequence GC content (%) of the target site.
Column 8: position The location of the target on the chromosome. The user can view it on the Genome Browser by clicking on it.
Column 9: genomic location The location of the gene at which the target is located.
Column 10: biotype Identifies whether the target is located in an intron or exon, or in an intergenic region.
Column 11: strand the strand of target sequence in the giving sequence. ("-" or "+")
Column 12: cutting effectiveness Identifies the on-target activity of the target, which is calculated by the SVM algorithm, and the higher the activity index, the easier the target is to cut.
Column 13: off-target risk Identifies the potential off-target risk of the target, the higher it is the more likely the target will be off-target and is not recommended.
Column 14: off-target detailDetailed off-target information for the target can be viewed by clicking on it.
Column 15: score Overall score of the target site. The target area, on-target activity and off-target risk are taken into account, with higher scores indicating a higher recommendation.
4. Description of off-target information
Off-target risk is formulated to compute the potential off-target sites associated with Cas9 RNA-guided endonucleases.
off-target detail information:
off-target detail information
Column 1: chromChromosome location of the off-target site.
Column 2: position Chromosome location of the off-target site
Column 3: strand the strand of off-target site sequence. ("-" or "+").
Column 4: sequence off-target site sequence. Mismatch sites are bolded in red.
Column 5: geneThe location of the gene at which the off-target site is located.
Column 6: location Identifies whether the off-target site is located in an intron or exon, or in an intergenic region.
5. Description of other functions
Results download: We provide downloads of the targets to facilitate the persistent use of these data by the user.
UCSC track download: We offer to download the results as track files for target visualization in the UCSC Genome Browser
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offline version: We provide an offline version of CRISPRlnc for download, with specific instructions and code available in github (https://github.com/Mera676/CRISPRlnc).