ZiFiT Targeter Version 4.2


Examples :

 

Zinc Finger Nucleases

CoDA (Context Dependent Assembly)

OPEN (Oligomerized Pool Engineering)

 

Example: CoDA (Context Dependent Assembly) - Nuclease

1) Select ZiFiT from the menu bar and follow the link labeled “Design Zinc Finger Nucleases” under CoDA(Context Dependent Assembly).


2) Paste the following sequence into the box labeled “Sequence” at the top of the input page. A case-sensitive search is denoted by a check in the "Exon/Intron Case Sensitivity" check box. Exons are denoted by uppercase letters. Introns are denoted by lowercase. Click the Submit button.

>IGF1R FASTA sample input

TTCCTTCCATTCCCTTGGACGTTCTTTCAGCATCGAACTCCTCTTCTCAGTTAATCGTGAAGTGGAACCCTCCCTCTCTG
CCCAACGGCAACCTGAGTTACTACATTGTGCGCTGGCAGCGGCAGCCTCAGGACGGCTACCTTTACCGGCACAATTACTG
CTCCAAAGgtaagggtgcagcagcggcctggacggagggtgtgaccgttcattcctgtggttgtaatgtgcctgagccct
aatattacacgtatcagacaacagtgtagttctccattggaaaccagctatcttctggttttttttttttttttttgaga
cagagtctcactctgttgcccaggctggagtgcagtggcacgatcatggctcactgcaatttctgcctcccaggttcaag
catttctcattccccagcattccgagtagctgggattacaggcatgcaccaccatgcccagctaatttttgtatttttag
tagagacggggtttcaccatgttgcccaggctggtctgaaactcctaacctcaggtgatttgcccgcctcggcctcccaa
agtgctgggattatagccttgagccaccacatctggccgagaccagctatcttcttgattaaaggtactgagagctatta
tttttccttacaagcatgtataacggctttcattcccactcttgttttggcttttcttttccgagaagACAAAATCCCCA
TCAGGAAGTATGCCGACGGCACCATCGACATTGAGGAGGTCACAGAGAACCCCAAGACTGAGGTGTGTGGTGGGGAGAAA
GGGCCTTGCTGCGCCTGCCCCAAAACTGAAGCCGAGAAGCAGGCCGAGAAGGAGGAGGCTGAATACCGCAAAGTCTTTGA
GAATTTCCTGCACAACTCCATCTTCGTGCCCAGgtacccagctcatgtgaaatttcagttggcaaaacccactgctcagg
ccggttctgttgcctttctccccaccaggtagtgtgtaagtcagcagctggggggtacaatacagtagccactgagacgg
agccgaaaaagatgacaggttcggtgaagtgagtccctgcggaagccagtcagccctgaagggaacagggaccagggctt
ggg


3) Graphic Summary popup (note popups must be enabled). Hits (blue, green, and gold bars) represent targets along the gene (red bars). ZFN hits in the graphic are color-coded based on spacer size (5bp=Blue; 6bp=Green; 7bp=Gold) and are linked to the detailed information in the main window.


4) Main Output Window (A - Condensed Target): Initially, individual hits are each displayed in their condensed forms consisting of a label, the double stranded target, and it's relative position within the submitted sequence. Any scores available for an given target are displayed on the same line as the target strand (No scores are currently available for CoDA). Potential targets can be sorted by position using the "Sort by" drop down menu. Targets can also be sorted by scores when available. Selecting the "Hide intron splice sites" to the left of the "Sort by" drop down menu will hide any ZFN Targets whose spacer falls outside of an exon. Individual targets can be expanded to display target-specific details by selecting the "+" to the left of the label.

 

5) 5) Main Output Window (B – Expanded Target): The expanded target view displays recognition helix sequences for the individual zinc finger domains used for generating CoDA proteins. The individual triplets are color-coded to match the corresponding triplet in the double-stranded target sequence. The “DNA sequence” hyper-link provides a popup with full length DNA sequences for these three finger arrays. Users can have the DNA sequence provided synthesized by a commercial provider. The sequence provided includes flanking XbaI/BamHI restriction sites (for ZFN pairs targeting sites with 5 or 6 bp spacers) or XbaI/NotI restriction sites (for ZFN pairs targeting sites with a 7 bp spacer. XbaI/BamHI or XbaI/NotI fragments can be cloned into plasmid pairs pMLM290/pMLM292 or pMLM800/pMLM802, respectively, to generate ZFN expression vectors (these plasmids are available through Addgene – see: http://www.addgene.org/zfc). For additional information on practicing the CoDA method, see Sander et al., Nature Methods 2011. Select the host genome for the ZFN target in the organism drop down box and select the "BLAST" button to scan the host genome for exact and similar matches. (Note: Users may need to wait for the ZiFiT output page to finish loading before using the BLAST features.)

 

6) NCBI BLAST Page: ZiFiT loads the parameters for scanning the host genome of choice to the NCBI BLAST servers. The nucleotides in the spacer have been replaced with N's to prevent them from positively influencing the scan. Select the "View Reports" button to initiate the query. Exact or extremly similar sites present potential points for off target cleavage. (Note: Due to the nature of the BLAST algorithm and the fixed spacer size, this query is not guaranteed to identify all similar sites.)  

 

 

Example: OPEN (Oligomerized Pool Engineering) - Nuclease

1) Select ZiFiT from the menu bar and follow the link labeled “Design Zinc Finger Nucleases” under OPEN(Oligomerized Pool Engineering).


2) Paste the following sequence into the box labeled “Sequence” at the top of the input page. A case-sensitive search is denoted by a check in the "Exon/Intron Case Sensitivity" check box. Exons are denoted by uppercase letters. Introns are denoted by lowercase. Click the Submit button.

>IGF1R FASTA sample input

TTCCTTCCATTCCCTTGGACGTTCTTTCAGCATCGAACTCCTCTTCTCAGTTAATCGTGAAGTGGAACCCTCCCTCTCTG
CCCAACGGCAACCTGAGTTACTACATTGTGCGCTGGCAGCGGCAGCCTCAGGACGGCTACCTTTACCGGCACAATTACTG
CTCCAAAGgtaagggtgcagcagcggcctggacggagggtgtgaccgttcattcctgtggttgtaatgtgcctgagccct
aatattacacgtatcagacaacagtgtagttctccattggaaaccagctatcttctggttttttttttttttttttgaga
cagagtctcactctgttgcccaggctggagtgcagtggcacgatcatggctcactgcaatttctgcctcccaggttcaag
catttctcattccccagcattccgagtagctgggattacaggcatgcaccaccatgcccagctaatttttgtatttttag
tagagacggggtttcaccatgttgcccaggctggtctgaaactcctaacctcaggtgatttgcccgcctcggcctcccaa
agtgctgggattatagccttgagccaccacatctggccgagaccagctatcttcttgattaaaggtactgagagctatta
tttttccttacaagcatgtataacggctttcattcccactcttgttttggcttttcttttccgagaagACAAAATCCCCA
TCAGGAAGTATGCCGACGGCACCATCGACATTGAGGAGGTCACAGAGAACCCCAAGACTGAGGTGTGTGGTGGGGAGAAA
GGGCCTTGCTGCGCCTGCCCCAAAACTGAAGCCGAGAAGCAGGCCGAGAAGGAGGAGGCTGAATACCGCAAAGTCTTTGA
GAATTTCCTGCACAACTCCATCTTCGTGCCCAGgtacccagctcatgtgaaatttcagttggcaaaacccactgctcagg
ccggttctgttgcctttctccccaccaggtagtgtgtaagtcagcagctggggggtacaatacagtagccactgagacgg
agccgaaaaagatgacaggttcggtgaagtgagtccctgcggaagccagtcagccctgaagggaacagggaccagggctt
ggg


3) Graphic Summary popup (note popups must be enabled). Hits (blue, green, and gold bars) represent targets along the gene (red bars). ZFN hits in the graphic are color-coded based on spacer size(5bp=Blue; 6bp=Green; 7bp=Gold) and are linked to the detailed information in the main window.


4) Main Output Window (A - Condensed Target): Initially, individual hits are each displayed in their condensed forms consisting of a label, the double stranded target, and it's relative position within the submitted sequence. Any scores available for an given target are displayed on the same line as the target strand. Potential targets can be sorted by position using the "Sort by" drop down menu. Targets can also be sorted by scores when available. Selecting the "Hide intron splice sites" to the left of the "Sort by" drop down menu will hide any ZFN Targets whose spacer falls outside of an exon. Individual targets can be expanded to display target specific details by selecting the "+" to the left of the label.

 

5) Main Output Window (B – Expanded Target): The expanded target view displays the required OPEN reagents for generating the libraries for each target. The reagents are color coded to the corresponding triplet in the double stranded target sequence. OPEN pools are available by request from the Joung lab (jjoung@partners.org). Other reagents are available through Addgene (http://www.addgene.org/zfc). For information practicing OPEN see Maeder et al., Molecular Cell 2008 and Maeder et al.,Nature Protocols 2009. Select the host genome for the ZFN target in the organism drop down box and select the "BLAST" button to scan the host genome for exact and similar matches. (Note: Users may need to wait for the ZiFiT output page to finish loading before using the BLAST features.)

 

6) NCBI BLAST Page: ZiFiT loads the parameters for scanning the host genome of choice to the NCBI BLAST servers. The nucleotides in the spacer have been replaced with N's to prevent them from positively influencing the scan. Select the "View Reports" button to initiate the query. Exact or extremly similar sites present potential points for off target cleavage. (Note: Due to the nature of the BLAST algorithm and the fixed spacer size, this query is not guaranteed to identify all similar sites.)