Webtool Usage

Run Parameters: DeepPrime

Recommended parameters:
1) For PE system, we recommend using PE2max with optimized scaffold in HEK293T cells as the initial analysis. According to our findings, it provides the best general prediction performance across diverse target sites. However, it can be adjusted to most closely match your experimental conditions.

2) Edit type and Edit length are constant at substitution and 1bp for saturated library design. For others, please run DeepPrime standalone webtool.

3) Length ranges for the reverse transcription template (RTT) and primer binding site (PBS) can be set to max defaults for maximum number of pegRNA considerations. The PBS ranges are set to a smaller for optimal processing times.

Run Parameters: Synonymous Mutation Marker

Synonymous mutation markers can be directed to a specific region of the target DNA corresponding to either the LHA, PAM sequence (positions 5 and 6), and the RHA. PegRNA designs that overhang onto intronic sequences can also be filtered out by toggling False.

Input Format:

GeneSym: Accepts common gene name or symbol. Ex) KRAS, BRCA1, ABL1, etc.
NMID: Accepts RefSeqGene IDs starting with "NM_##.." from NCBI database
Ensembl ID: Accepts Ensembl IDs starting with "ENSG##.." from Ensembl database
HGNC ID: Accepts HGNC IDs starting with "HGNC:##.." from HGNC database
ClinVar ID: Accepts Variation IDs or Accession starting with "VCV##.." from ClinVar database
COSMIC ID: Accepts Mutation IDs starting with "COSM##.." from COSMIC database
*Note: Full reference gene list can be downloaded using the Support drop-down menu or here

Sequence input accepts a wild-type (unedited) and edited sequence of 121bp in size, 60bp sequences flanking the 1bp substitution. Example Position input accepts common genomic coordinate format of chr#:####-####. Coordinate can be continuous or a single position. Ex) chr17:7675207-7675209

Results:

The interactive results section allows users to examine the overall coverage of their target gene exons with a visual representation of the unique pegRNAs for each edit position. Additionally, the top pegRNA designs can be examined with their features of the pegRNA and pre-determined oligonucleotide sequences for library construction. In addition, the predicted efficiency score is normalized using rank and compared to other pegRNAs used in model testing for the corresponded PE system. The full results with comprehensive details of all pegRNAs can be downloaded using the link at the bottom of the Results page.

Example Result Pages:
GeneSym Example: TP53 - Exon 5
GeneSym Example: BRCA1 - Exon 3
ClinVar Example: 1714092 (KRAS)
COSMIC Example: COSM4047104 (BRCA2)