E-CRISP Design

Indicate which reference genome E-CRISP should find targets in?
Indices are pre-built for each organisms individual gene sequences

Enter either:

a ";" separated list of official gene symbols or Accession IDs. For e.g.

PFKP;PFKFB3;PRKCQ;CAMK1D (for Human)
ENSDARG00000090752;ENSDARG00000063570;ENSDARG00000074914;ENSDARG00000032801 (for Zebrafish)
FBgn0000442;FBgn0250906;FBgn0020618;FBgn0044323 (for Fly)

OR

one or more Fasta formatted sequence. For e.g:

>sequenceID
ATATCGATGCTAGCTAGCT
GATCGTAGCTAGCTAGCTA

Transcript IDs (ENST...) can be entered in subsection 4
but still require the respective gene to be entered here.

Maximum length of the sgRNA target site (protospacer)

Minimum length of the sgRNA target site (protospacer)

Adjust the length of the sequence that will be saved 3' to the cut site.

Adjust the length of the sequence that will be saved 5' to the cut site.

TTTT motifs can hinder RNA transcription. Thus it can be wise to exclude target sites harboring such motifs.

Target only inside the gene of interest.
Else designs can also be up to 500 bp up-/or downstream of the desired gene.

Check if designs should only target exons.

Check if designs should target only outside of predicted CpG islands.
(Outside potential hypermethylated regions)

Enter a specific ensembl transcript ID ENSTR.......

Enter the exon number to target. E.g. Enter 1 to find designs which target the first exon exon.
Or enter the word any to find designs that hit any exon.

Check this box to assess restricion enzyme cut sites in the whole target sequence.

Choose between different pre-defined restriction enzyme subsets.

Leave this box checked if designs should be analysed for targets in the reference genome.

Should the CRISPR design be checked for any secondary off-target effects?
This is useful to check if they will cut in any exogenous, foreign introduced sequence.
Check every individual off-target or paste a set of fasta sequences into the text area

This text area only accepts FASTA format sequences as input e.g.:

>some sequence
ATATCGATGCTAGCTAGCT
GATCGTAGCTAGCTAGCTA

E-CRISP
Design of CRISPR constructs

DKFZ

Check out our new CRISPR Library Designer (CLD): batch design of sgRNA libraries Download the dockerized version now at CLD on Github
1. Select organism:
[HELP]

2. Select target region by gene symbol or sequence:
Input is GeneSymbol
Input is FASTA sequence
TP53 FASTA example \| GeneSymbol example \| Clear [HELP]
case-sensitive search in ENSEBML Ids search in gene symbols search sensitivity: lowhigh	Search results (click to add, scroll down to see full list) Search results (click to remove, scroll down to see full list)

3. Start application:
relaxed (any PAM (NAG/NGG...), any 5' base (A,C,G,T,...), off-targets need full length perfect match, introns are allowed) medium (any PAM (NAG/NGG...), any 5' base (A,C,G,T,...), off-targets tolerate mismatches, introns/CPG islands are excluded) strict (only NGG PAM, only G as 5' base, off-target tolerates many mismatches and ignores non-seed region, introns, purpose is knockout (only first 3 coding exons are allowed) and UTRs are excluded)


5. Design purpose:
Select if CRISPR designs should be used for tagging or knockout experiments
bp Minimum guide RNA length after PAM [HELP] bp Maximum guide RNA length after PAM [HELP] % < G < % % < A < % % < T < % % < C < % bp 3' flanking sequence length [HELP] bp 5' flanking sequence length [HELP] bp CRISPRi downstream window bp CRISPRi upstream window bp CRISPRa downstream window bp CRISPRa upstream window	bp Tagging window downstream of the codon bp Tagging window upstream of the codon Number of coding exons downstream the start codon for K.O. bp Minimal spacer length for paired designs bp Maximum spacer length for paired designs 5' preceding Base requirement 3' PAM IUPAC basepair code (click me) custom PAM sequence (3'PAM) exclude targets with poly T motif[HELP] exclude targets with poly A motif exclude targets with poly C motif exclude targets with poly G motif Number of bases in the homopolymer

6. Gene annotation filtering:
Exclude hits outside the gene [HELP] Exclude designs in overlapping genes Exclude non-exon hits [HELP] Exclude hits outside of coding sequences Exclude hits in CpG islands [HELP] Transcript specificity [HELP] Exon specificity [HELP]	Retrieve and save a recombination donor matrix Assess restriction sites of the whole sequence [HELP] Restriction enzyme library [HELP]

7. Off-target analysis:
select bowtie version select off-target database Exclude designs with more than X off-targets Number of 5' mismatch positions Tolerated edit distance to the target sequence check off-targets in non-gene regions should be ignored Bowtie2 pre-sets Analyse Off-targets [HELP]	Select to check for secondary off-targets [HELP] Please choose the targets to test and/or paste a custom sequence into the text area below. The pasted sequence should be FASTA format. [HELP]

8. Output:
Maximum number of results per exon Produce a GFF formatted output file Create an image showing genomic context Add restriction sites to the image Add TSS to the image Add stop codons to the image Add start codons to the image Output the result table to the browser window Produce additional information for the Matchstring (Warning: This option might take a while!) should E-CRISP continue processing if input IDs cannot be found

The older version of E-CRISP can be reached Here

Boutros lab, E-CRISP-Version 5.4
For suggestions please contact us at crispr@dkfz.de