Using SynDesign

Using SynDesign pegRNA_schematic

Load Example

The Load Example button is able to replicate the example inputs shown in this help page for quick testing of the SynDesign web server. Simply click the Load Example button then press Submit Job to obtain an example results page.Parameters

Prime Editor system: Choose model conditions closest to experimental parameters. We recommend using the model PE2max with optimized scaffold in HEK293T cells for the initial analysis. According to our findings, it provides the best general prediction performance across diverse target sites.

Edit type and Edit length can be adjusted according to the desired edit, and these values are only available in the Edit Sequence input format; a Genomic Position run designs a single-base substitution at every position in the range. Note that any value other than Substitution with length 1 may lead to much longer processing times due to the increased number of possible pegRNA designs, as well as the possibility of failure or timeout.

Primer binding site (PBS) and reverse transcription template (RTT) lengths can be set to the maximum ranges (1-17 for PBS, up to 40 for RTT) for the maximum number of pegRNA considerations. However, the ranges can be adjusted to be narrower for faster processing times, and we recommend the default PBS range of 7-15 for optimal balance.

Silent Marker Priority:

Choose the priority for silent markers between the left homology arm (LHA), a PAM edit and the right homology arm (RHA): Default, LHA only, PAM edit only, or RHA only.Schematic of selected region(s) for inducing synonymous mutation markers:

Left Homology Arm – LHA:

PAM edits inducing PAM disruption:

Right Homology Arm – RHA:

A target near an exon boundary puts part of this window outside the coding sequence. Intronic bases belong to no codon, so there is no synonymous change to make in them and a marker can only be placed in the coding part: pegRNA_schematic_intron

The pegRNA itself is unaffected — it is designed from the coordinates either way. It is the synonymous marker, and only the marker, that needs a reading frame.

Top Guides for Display: Select the number of top guides to display in results. Default: 200.

Input FormatsSynDesign supports multiple input formats: Input_FASTA

FASTA: Upload or enter FASTA sequence(s) to perform saturation genome editing (SGE) on the provided sequence(s). The recommended sequence length is >= 121 bp, where a 60 bp flank on either side of the target site gives the fullest set of pegRNA designs; a shorter sequence is designed from and offers fewer.

When uploading files, press the [+] button to add files. Uploaded files can be cleared by pressing either of the checkmark buttons. Input_Data

GeneSym: Gene Symbol (e.g., BRCA1) and Exon Number.

NMID: RefSeq NM ID and Exon Number.

Ensembl: Ensembl Gene ID and Exon Number.

HGNC: HGNC ID and Exon Number.

ClinVar: ClinVar ID and Analysis Mode (Model/Correction). Use the numeric Variation ID (e.g., 9325), the VCV accession (e.g., VCV000009325.76), a ClinVar record (RCV) or submission (SCV) accession, or a dbSNP rs number (e.g., rs80357382). An RCV or SCV accession names an interpretation or a submission rather than the variant, and its digits are not the Variation ID, so it is looked up in the bundled ClinVar release and translated to the Variation ID behind it, with the translation stated in the job log; one the bundled release does not carry is refused, with the reason.

COSMIC: COSMIC ID and Analysis Mode (Model/Correction).

Sequence: Custom Unedited and Edited Sequences. The edit is read from the difference between the pair, wherever it sits in them. A 121 bp sequence — 60 bp of flank on each side of the edit — offers the fullest set of pegRNAs, because it is the shortest input in which every candidate's scoring window fits; a shorter pair is designed from and offers fewer candidates. No pair is skipped for its length.

Position: Genomic Position for GRCh38 (chr & coordinates).

Note: GeneSym, NMID, Ensembl, HGNC, and Position inputs may take dozens of minutes to hours for large inputs. Long sequences in FASTA mode will also similarly increase processing time.A list of available gene symbols and annotation IDs can be found in this Text File. Load_Example

Examples for each mode can be loaded by (1) selecting the desired input format, then (2) pressing the Load Example button.SpeciesA gene identifier is looked up in Human (GENCODE v50 on GRCh38) or Mouse (GENCODE M39 on GRCm39). A mouse symbol from an older nomenclature resolves to the current one and the job log states the substitution — Olfr1 designs against Or1e16. Whichever of the four gene formats is chosen, Mouse replaces the identifier box with Enter Mouse Gene Name or ID, which takes a mouse symbol or a mouse Ensembl gene ID. ClinVar ID and COSMIC ID carry no species selector: both are human variant databases, and both resolve against the human reference.A mouse gene is resolved to its coding exons and the design runs across those, so the pegRNAs are the same work the human path does. What mouse does not carry is the coding-position summary, which comes from the human gene records rather than from the design.Off-targetNo off-target control is offered here, and the results carry no off-target column: there is no reference genome to choose, no genome-wide search, and no mismatch-tolerance score from a substitution table. Prime editors are far less sensitive to off-target editing than nucleases are — installing an edit needs the spacer to match, the nick to be made, the extension to anneal and its template to be copied in, where a nuclease needs the spacer alone — so the sites a counted-mismatch genome search returns are not the sites a pegRNA edits. The one trained prime-editing off-target model is DeepPrime's, and it is a separate output on the DeepPrime workflow.Engineered pegRNA (epegRNA)pegRNA Form decides the construct and is set to pegRNA (Unmodified 3' End) by default. Setting it to epegRNA appends a 3' structured motif to the extension and adds one orderable oligo pair per motif to the results table. The motif protects the pegRNA 3' end from exonucleolytic degradation. An epegRNA is a different construct to order from a pegRNA, which is why the motif is asked for rather than applied by default, and epegRNA Motif Panel — shown only while epegRNA is selected — chooses which motifs: the Recommended Panel (eHAV, tevo2.0, HAV), the Extended Panel (those three plus eSBRMV1-A, FMDVA-2 and tevopreQ1), Synthetic / LNP (eSBRMV1-A), the v5 Library Screen Panel (eHAV, tevo2.0, HAV, eSBRMV1-A, eFMDVA-2.2 and eFMDVO-4), or any single motif on its own. The panel is offered rather than ranked because no feature predicts which motif wins.No linker is inserted between the primer binding site (PBS) and the motif, and that is deliberate rather than an omission. The v2 motifs offered here (tevo2.0, eHAV, HAV, eSBRMV1-A and the FMDV motifs) are used by Sakai et al. with the motif joined directly to the PBS 3' end; their assembled-construct tables carry no linker field. For tevopreQ1, Nelson et al. compared their pegLIT-designed 8-nucleotide linkers against no linker at all and reported no significant gain in editing efficiency (Supplementary Fig. 13). The one motif their data shows does need a linker is mpknot, which is not offered here.If you are building an mpknot epegRNA elsewhere, design its linker with pegLIT (https://peglit.liugroup.us) rather than reusing these oligos.Oligo OutputCloning Vector decides whether each candidate also carries an ordering-ready spacer oligo pair, and it is set to SpCas9 / lentiGuide-Puro (BbsI or BsmBI) by default. The pair arrives as Oligo (forward) and Oligo (reverse) in the results table and in the downloadable files alike: the two strands that anneal into the digested vector, CACC-presenting on the forward strand and AAAC-presenting on the reverse. Setting the vector to None (No Oligo Output) leaves both columns out rather than blank, and the four overhang fields override what the vector supplies.Results Result_Summary

The first part of the results page is a summary of the pegRNAs generated for the input sequence or gene, including the total number of pegRNAs and the coverage of the gene. Result_Table

The second part of the results page is a list of the top guides for display, sorted by the DeepPrime pegRNA score. All columns may be sorted and filtered in the table. Result_Details

Selecting each pegRNA entry on the table will update the Selected pegRNA Details and pegRNA Oligo Design sections, identifying various characteristics of the pegRNA, including the sequence, oligo design, and predicted efficiency. Detail_Fig

The above figure outlines the relative efficiency of the pegRNA compared to all pegRNAs generated for the input sequence or gene. Download_Results

The Download Results button will download a ZIP file containing all pegRNAs and details outlined above.The archive also carries the design engine's own dumps, the files ending _fullout.csv, _pegRNAIDs.csv and _oligos.csv. Each keeps the engine's own columns and is keyed by design id rather than by a target window, so there is nothing in it for a derived column to be placed against, and the oligo dump's own pairs are the engine's fixed overhangs. The derived columns for the options a job submitted — the spacer and extension oligos for the chosen cloning vector, the epegRNA extensions, and everything else described above — are on output.csv and summary.csv, and on the archive's own copies of those two tables, which it carries under the engine's names for them, Results_forHTML.csv and Summary_forHTML.csv.GlossarySequence and Design Components

Spacer (Top/Bottom): The 19-nt guide sequence of the pegRNA that determines the specific genomic target for the Cas9 nickase, as the annealed oligo pair to order. Cloning Vector decides whether the pair is built at all — set it to None (No Oligo Output) and those columns are left out rather than blank — and the four overhang fields beside it (Forward 5′, Reverse 5′, Forward 3′, Reverse 3′) override the vector's own CACC/AAAC, each field left blank taking the vector's own.

Extension (Top/Bottom): The 3' extension of the pegRNA that contains the PBS and RTT sequences necessary for reverse transcription, as its own oligo pair. The four Extension overhang fields override its GTGC/AAAA the same way.

WT Sequence 74bp: The "Wild Type" or unedited genomic target sequence, represented in these studies as a 74-nt sequence used to identify the target and evaluate prime editing outcomes.

Edited Sequence (74bp/with Note): The desired genomic sequence after the intended prime editing modification has been successfully integrated.

Guide Sequence: The 19-nt sequence within the pegRNA that directs the prime editor to a specific DNA target.

PBS Length: The sequence in the pegRNA extension that binds to the nicked DNA strand to initiate reverse transcription.

RTT Length: The part of the pegRNA extension carrying the desired edit and the homology sequence required to integrate it into the genome.

RHA Length: The rightmost portion of the RTT sequence that provides homology to the genomic DNA after the edit position.

PBS-RTT (Reverse Complement): The total combined sequence of the PBS and the RTT. Due to matching the target sequence instead of the pegRNA, the actual oligonucleotide would be the reverse complement of this sequence.

Scoring and Statistical Metrics

DeepPrime Score: A predicted efficiency score generated by the DeepPrime deep-learning model (convolutional neural network + gated recurrent unit) indicating the likelihood of a specific pegRNA successfully inducing the desired edit.

Z-Score: A statistical measure used in SynDesign to depict how the predicted efficiency score of a specific pegRNA compares to the distribution of scores from model testing under identical prime editor and cellular conditions.

Percentile: The relative rank of a pegRNA's predicted efficiency compared to all other feasible designs for that specific target.

Genomic and Edit Context

Edit Position: The location of the intended mutation relative to the Cas9 nick site, with the nucleotide immediately after the nick designated as position +1.

Genomic Position: The specific coordinates of the target gene or variant within a reference genome, such as GRCh38/hg38.

Synonymous Mutation Locale/Marker: A silent mutation (one that does not change the amino acid) introduced near the intended edit to increase efficiency by disrupting mismatch repair (MMR) and to serve as a marker for identifying edited products during deep sequencing.

DNA Change: The specific nucleotide alteration introduced into the genomic DNA.

AA Change: The corresponding change in the protein's amino acid sequence resulting from the DNA modification.

Sample OutputsThe following example interactive outputs are available for reference:FASTA sequenceGene symbolClinVar variantSequence pairGenomic coordinatesContactIf you have any questions, bug reports, or suggestions, please do not hesitate to contact us through the Support page.

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