Revvity’s Pin-point™ base editing design tool makes it simple to create base editing sgRNAs directed to splice donor, splice acceptor, or premature stop codon sites for targeted protein knockout.
Please note that the custom Pin-point sgRNAs designed with the Pin-point base editing design tool are not functionally guaranteed.
Enter a human Gene ID or Gene Name
The Gene ID is the unique ID number assigned to each gene in the NCBI database, as you can find, for instance, on our website (see example list below). Do not hit Enter when you type or paste your gene into the search. You must wait and select the gene from the drop-down menu. Then click on "Build Custom Pin-point designs".
672 (BRCA1)
25 (ABL1)
983 (CDC2)
6419 (SETMAR)
Advanced Options
Optimal Base Editing window:
C2-C9: Select to show additional candidate guides that may have a lower potential for achieving optimal base editing activity.
C4-C8: This is the default setting as it is the optimal activity window for Rat Apobec in the Pin-point base editing configuration.
Base Change Type:
Splice Acceptor: Design sgRNAs to interrupt a splice acceptor site for protein knockout. Site is generally: intron-AG|exon with a target “C” edited on the (-) DNA strand.
Splice Donor: Design sgRNAs to interrupt a splice donor site for protein knockout. Site is generally exon|GT-intron with a target “C” edited on the (-) DNA strand.
Premature Stop Codon: Design sgRNAs with NGG PAMs that allow the conversion of CAA, CAG, CGA, or TGG codons into stop codons for protein knockout.
Accession Targeting: Accession numbers can be found in the NCBI Reference Sequence (RefSeq) database. This is a non-redundant, curated, and annotated collection of sequence records for major model organisms.
Select the accession numbers that you wish to visualize. Please note that this does not mean unchecked accession numbers won’t be targeted. It means that unless selected, that accession number will not be visualized in the results list.
RefSeq accession numbers use the systematic naming convention below.
| Accession Prefix | Molecule Type | Description Notes |
|---|---|---|
| NM_ | mRNA | Protein-coding transcripts. Usually curated |
| XR_ | mRNA | Computationally predicted model protein-coding transcripts |
| NR_ | RNA | Non-protein-coding transcripts |
| XR_ | RNA | Computationally predicted model non-protein-coding transcripts |
Results
A list of the potential sgRNA candidates is presented along with details which will help you filter and select the best sgRNAs.
Using the Advanced Options on the results page, you can filter the results to show only the desired editing window, type of change and accession number(s). However, filtering is only for visualization. It does not mean that a specific guide will not target unselected options.
Target sequence: Guide targeting sequence with cytosine base(s) falling within the selected base editing window marked in red (C).
Specificity: Specificity score assigned based on the likelihood of the target sequence and associated complementary mismatches occurring elsewhere in the genome. A higher specificity indicates a lower likelihood of potential targeting elsewhere in the genome.
Genomic Location (Strand): The specific region in the genome that the target sequence is directed to (hg38). (+) denotes the positive strand and (-) denotes the negative strand.
Targeted Region (Target base): The RefSeq accession number, exon, and cytosine base position within the spacer targeted to create the desired change.
Type of change: Splice site disruption or stop codon creation with the specific amino acid change created listed as either "Introduce Stop Codon" if a new stop codon is introduced, or "Replace Stop Codon" indicated if the C to T change results in one stop codon being substituted for another.
% distance from CDS start*: Position of sgRNA relative to transcription starting site. Give higher priority to guides located within the middle 10-60% of your transcript.
CDS length*: Coding DNA Sequence length.
*Splice acceptor and donor changes return an N/A for % distance from CDS start or CDS length because the splice sites do not actually sit in a CDS. They sit at the junction between coding sequences.
PAM: Protospacer adjacent motif (PAM) sequence. A short DNA sequence is required for Cas enzymes to recognize the target site. The Pin-point nCas9 uses NGG PAM and it is always located at the 3' of the spacer on the targeted strand.
Disclaimer
All custom sgRNA sequences must be experimentally validated and may require obtaining additional licenses by the end-users. Specificity of sgRNA sequences should always be considered to reduce off-target effects.
By accessing or using this site, you acknowledge and consent to the following:
The use of this site is provided free of charge to the research community for basic research and educational purposes only. Use of the site and of custom sgRNA sequences created with the site are for research use only. No other use is permitted.
This site and the custom sgRNA sequences created by using the site are provided on an "as is" and "as available" basis without any warranties of any kind, either express or implied, including, but not limited to, warranties of merchantability, fitness for a particular purpose, or non-infringement. We make no representations or warranties that the custom sgRNA sequences will not infringe upon the intellectual property rights of any third party, including patents, copyrights, or other proprietary rights. To the fullest extent permitted by law, we shall not be liable for any incidental, consequential, or indirect damages, including but not limited to economic loss or personal injury, arising out of or in connection with the use of the custom sgRNA sequences.