NGEC Targeted Gene Sequences

Canine XSCID exon 1 with underlined deletion present in the canine XSCID model:

ATGTTGAAGCCACCATTGCCACTCAGATCCCTCTTATTCCTGCAGCTGTCTCTGCTGGGGGTGGGGCT GAACTCCACGGTCCCCATGCCCAATGGGAATGAAGACATCACACCTG

I realize this is only 100 base pairs, but if you can target within this somehow, that would be by far the best. Below is a longer sequence of canine genomic DNA, which includes about 200 base pairs of additional Upstream sequence, and 400 or so of downstream sequence. Note deletion is underlined, so these base pairs will not be present in the gene which needs to be cut.

CGGAAACTATGACAGAAGGAAATGTGTGGGTGGGGAGGGGTAATGGGTGAGGGGCCCAGGTTCCT GACAGTCTACACCCAGGGAACGAAGAGCAAGCGCCATGTTGAAGCCACCATTGCCACTCAGATCCC TCTTATTCCTGCAGCTGTCTCTGCTGGGGGTGGGGCTGAACTCCACGGTCCCCATGCCCAATGGGAA TGAAGACATCACACCTGGTGGGAAACATGGGACTGGAAGGGGTTGGTGAGAGGGGAGCCTGTGGG AAGGGGTCGCATAGAAATCTTGAACCTGCCATGGGGCATTAGAAGGATGTGGGCAGAGTTTAAGAG TGCTGTGGAGAAGGAAGGGGGAAGGGGTTCACTGCCTCATGCTGCTGCTTCTTCTGACCATCATTTC TGGTTTTTCCCTCCCCCTCTTCATTTTCTCCACAATCTAGATTTCTTCCTGACCGCTACACCCTCCGAG ACCCTCAGTGTTTCCTCCCTGCCCCTCCCAGAGGTCCAGTGTTTTGTGTTCAATGTTGAGTACATGAA TTGCACTTGGAACAGCAGCTCTGAGCCCCGGCCCACCAACCTGACCCTGCACTACTG

Murine XID - exon 2 contains the mutation:

Exon 2 is: TACACAAACAAGTTCCAGAGAGAGGAAGCCATGGCTGCAGTGATACTGGAGAGCATCTTTCTGAAG CGCTCCCAGCAGAAAAAGAAAACATCACCTTTAAACTTCAAGAAGCGCCTGTTTCTCTTGACTGTAC ACAAACTTTCATACTATGAATATGACTTTGAACGTGGG

The above exon with surrounding sequence is:

GTGTAAAGTGGTGTCTAGACATTACTGACCACCCAGGGCTATGTTGTCGGGCTCTAGAGCATTCAGTTCCCTT ACCCAAGAAGCCCAGTTTTGTAAACTCACCATGTAGAATCAATCCCCTGGTTAAATAGTATGAGTTTGGGCTG GAGAGAAGGCTCAGTGGTTAAGAGCACTGACTGCTCTTCCACAGGTCCTGAGTTCAATTCCCAGCAACCACAT GGTGGCTCACAGCCATCTGTAAAGGGATCCGATGCCCTCTTGCGGCATGCAGGTGTACATGCAGATAGAGCAC TCACACATAAAATAAATAAATCTTTTTAAAAAAAATAGTATGCGTTCACTGTACTTGAAATTTGACCAAGATG GGGGGACTGTGGAAGAAGGAGCTCAGGCAGAAAGAAACAAGATTCCCTGCTAGCGTTGTATATTTGTGTTTT GTTTACAACAGGGAGCAGGGCTGAGGCAGAACCAGGTAGGACAAAGATGACTATGCTAGAACCTTAAAAACT TTCCTTATCCACAGTACACAAACAAGTTCCAGAGAGAGGAAGCCATGGCTGCAGTGATACTGGAGAGCATCTT TCTGAAGCGCTCCCAGCAGAAAAAGAAAACATCACCTTTAAACTTCAAGAAGCGCCTGTTTCTCTTGACTGTA CACAAACTTTCATACTATGAATATGACTTTGAACGTGGGGTAAGTTTCCCAAATGAGAAGTCCAAATTTCACC ATAGTCTTGGATCTTCTGTGAGAAATGAGATATGTGGTAGGTAGGAACTTAGTAGAGCTAAGTACATCAAACT TGAAAACATATTTCCTAGCCTAGGAGTAGGCACAGTCATGGCAGGGGAAAAGTCAAATATGTTAAGAAGCTG TTCAGAATGTCTTACCATGGAATTGTGTGGTTAGTAAGAACTCACTCTATGCTTCTTGAGAATGAAATCCAAG AATTTGACATTGCGTTTTTATTTCTGGAACAATAATGCATATAATCATTTATTCTTCTATGTTATCTGCTTCCCA TTTGGCATTTAAAGGTTAAATAAAATGCTGGATTTGTTTAAAACTAACAGGTTCTGCCAGGCACAGTGGCACA TACTTTTAGTCCCGGCACTCAGGTTTACACAGTGAGTTCTAGGCCAGCCAGGGCTACATTGTGAGACCTTGAG TCA

The mutation is an R28C, with CGC in the following sequence changed to TGC: GAAGCGCCTG to: GAAGTGCCTG Canine PK deficiency:

Below are intron 4-exon5-intron5 of canine PK, including the deletion mutation underlined and bold.

GTTTGGGGCGGGGCCAAGGGGAGGCTGGCGAGCTAAAGCTTGCCGGGTTGAGAGGTGGGGGGCCG GGCAACCTGCTCCTGAAATCCCCTCTCGCTGGTCCCCAGTACCATGCTCAGTCCATCGCCAACATCC GGGAGGCCGTGGAGAGCTTTGCAACTTCTCCGCTCGGCTATCGCCCCGTGGCCATTGCCCTGGACAC CAAGGGACCGGAGATACGGACCGGGGTCCTGAAGGGGGTGAGAGGCCAACTACGCCCGGGGGCTT CCCTAGGCCCCCACCACCCTGTCAGGGACTGAGTCGCGGGTGGGGGCGGGGCCTGGGAGGGCAGGG CAAGAGGCGGGTCTCAAGGCCCCTCTGGCTGGTCCCTAGG

FAH murine model in Grompe Pilot project: mutant mouse ideal target sequence:

GCTTTCTTCGTAGGCCCTGGGAACAGATTCGGAGAGCCAATCCCCATTTCCAAAGCCCATGAACACA TTTTCGGGATGGTCCTCATGAACGACTGGAGCAGTAATGCCTGGTGGCCCAGCTTCCTCTGATGTTCT GTTCTTAGGGGCACACACAGGAGTTGGGTATGGGACAGGAGGCCTAAGTACTACAGGGGTGATACC ATGC

Note underlined/bold A: In the WT mouse this is a G, and it is the terminal codon of exon 8. In the FAH5961SB mouse model of tyrosinemia, this is an A, as above.

Markus' strategy utilizes a 4500 base pair region of homology delivered by an AAV vector, so it may be that it would be interesting to target farther away, and look at the repair efficiency. Therefore, while it is best to try to get something in the above sequence within 100 base pairs or so of the target, if you can find an easy target in the following longer sequence, that would be worth going after as well.

(sequence from Andy – 3150 bp – genomic w/ intron) AGACATGGAGTTGGAAATGGTGAGTTCTGTGTGGAATTTTGTTGAATGGGATCTACAAAGCGCTGTGGCAGTA AAGCTCTCTTTCTGGGGCGGCACTTCACACCTGTTTGGCCACAAATAGCAGCCCATGTCCCCATGTGACCACT GAATGCAGTTGATGTTCTTTCTTCAGATTCTGTGTTTGTGAATTCTCCCACTTGCTGAAACTTATTTGCAACCCA AATATTAACACAAGTCATTGCCACACAGAGAAGTGGAAAATTTGAGTCTCCTGATACACACATTTGTCCCCAG TTGAGGTCACACCAGGCTCCCTGTCTTCGTGTTTCAGGTCTCATGCTATAAAAACATGCTATATTTTTGGTTTTT TTTCTCCACATATTTTCCACATTTGAACTACTTTTATTATTTACAATGCCTCCCAAGTGGAGGTCCATTGTGGTT CCCTAACTGCAGGAGGGCAATTCTAACAAGTGTTAGAAAAGCCTCATTCATACCTAAGTTGTAGCTGTTGTTG GGGAATACAGTGTTGTTGAGCCAACACTATGTCAGCTATCTTTAACAGAAAGAGGTAAAATATGGTGAGTGTT GTAGCCAGAGACTATGAAAACCCAGCTTGTTGCATCTTGTGGGAGCACCGGTCCACCTTTTTTCTGTCACATTC ATGGTGATTTTATACAATGTCACATGAACAGCAAGCATCAAGGCCCGGTGGCAGGGCCTCTATCACTTTAGAA AGACCCACACTAAGGCTCCACTCTTTATTTAAATTAAGCACTTTATTTTGAGAGTGCTGAAGACTCATATGTAG TTGTGAGAAATGAGGTGGAGATCCCCTGAATTTGAAAGAGATGGGGTCCTTCATGCCTCGGTGCCCTCAGCTC TCTCTCTCTCTCTCTCTCTCTCTCCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTGTGTGTGTGTGTGTGTGTGTGT GTGTGTGTGTGTGTATCTTGAAGATTTTGCACTGTTTAAACCCTATCTCAAGGACAAATAAAACAGTGACCCT AGCCATGAACGTAGATGATTCTGTACTGCTCAAGGAATTCCTTCTCCCCCGCACTTAGTTTCCCTTAGACATGA TTGCTAGATGTTCTCTTGGTCCATGGCTATTGGACAGATGCCATTCCTTTTGGGATAGGTAGGGCTGTGAAGTC AACTTGTCAATCCTCCTGGAATGAGCACCCCTGATGTTTTCATTCTTTACAAGTGTCATATACTGTCAGATGTG AGGGTATGGAGCCTGGAATACAGAGGAAGAATCAGAAAGGCCTGGGGTGCCCTGGGGCTAGGGGGGAAAGG GCTCATGTGAGTAGACTTCCCACCTTAGAGACCCAGGGAAGTAATGCCAGGTCCTCAGGCAGCCCTAGTCCCT GGTTGAACTTTGAAAATATTTTCCCTTTGCTCTGTAAGCCACAGTGACCCAGAGCATCGGGTCATCTAGATTCT TACCAACTTTCTCCATGGCAGGCTTTCTTCGTAGGCCCTGGGAACAGATTCGGAGAGCCAATCCCCATTTCCA AAGCCCATGAACACATTTTCGGGATGGTCCTCATGAACGACTGGAGCGGTAATGCCTGGTGGCCCAGCTTCCT CTGATGTTCTGTTCTTAGGGGCACACACAGGAGTTGGGTATGGGACAGGAGGCCTAAGTACTACAGGGGTGA TACCATGCAGACTTCTGACTCTGTGGGTGTGGGGCAGTCACAGCTTCCCTGAGTAGCTTTCTCATAAGTGGAA GGATGGAGCTGACAGAACCTAAAGCTTTATCAAGCCCTACACACTCCACTCACTGTTGCCAGCACCATCAGCT GCTGATGAAGAGCTGTACATGGAGGGATTTTAGTCAGTGGCTGAATTGAGCCTTACTATTCATCAGCCCAGCA TCTGAGTCCCAGCTCTACACCCTGATACCATGTGTAAGAGGAAGTGGGTCCAGAAATCTCAGGAGAGGGCCTT CCCCCATTCAGGCCCTTGGTAGGGACTGTCCTGTCATCCTTCATTGTTCCCTAGCACAGCACCTCCAAGGACCT CTGTCTTCATACTGAATACCTTTCTGTTTGTTCCCTGGGAGATCTGGTTGGCCAGAGCCATGTAGAGAGGTCCA TGTAGGTCAAGGACAGGAAGCAGCTTGGAGTGAGTCTGTAAGCCACGGTGAGACCTAAGCTTGCTGTCTTTTC ATAGCACGAGACATCCAGCAATGGGAGTACGTCCCACTTGGGCCATTCCTGGGGAAAAGCTTTGGAACCACA ATCTCCCCGTGGGTGGTGCCTATGGATGCCCTCATGCCCTTTGTGGTGCCAAACCCAAAGCAGGTAAGCACCC TTCTGCTGAAAACTCTGCTCTAAGTTCCGGCTTCCGCCCTTTCCTGTTGCCCTCTGATACTGAGACATGGTGAT GCTTGGTGGATCCTGCATTTTGTTTTGTGCAATGGCTCCCTCTAGAGTCTAATTTGCAGAATGCATTTTGGAAT AGAGGCTATAAGGGAAAAGGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTCCAACA GGGTTCCCAGTGCAATATCCCACCCCTTCATTACAGTCACCCAGCTCTTATGGCCAATGCCTTGAAGGCCCTAA CTTCTCTCAAACACGGCTCTGGTAAATGACCTATCTGGTCCTCCCACATCTCTGGGTCTAAATCTCCAGAGGGC TCTGCTCTGCTCCTGGCTCCCTGCCTTCCCTCCCCACACTCTCCCTGGCATTGTCTCACCAGCTGTGTACTGCTG CACTTTCTCCCTCACCCATCCATGTCATTTTATGCATGAACACTCTTGGGACTTTCCCCTGCAAATTGGTTCTTC ATAAAAGTCCCTGCAGTTCCATTCTGTCTCCTTGGATCTTCCCCAACACTTCCCACCACAGCACCCACGTCTCC CCTCTGGTGGTCTCCCTCCTCTTTGTCTCTGTCCCTCCACTGTCCTTCTCATCTTCTGGCCACCCAGGACCTGTT GGGCTCCTGCTTTAATGGTCCCCATCCTCCTTCACAGTCCTTCGCCCAGCTCTATCTTCACAGAGATGATTGGA TGCTGACTGCCTCCATCCTTCCCAAGGACTGTTTAAGTACAGCTGTCACCATTCCTGC

Primate (Macacaque) CCR5: We simply want to disrupt this gene, so can target anywhere.

> NAME = 'nemestrina promotor and UTR.fa' : TYPE = DNA GCACAGGGTTAACCCGAAATCCAGGATCCCCCTCTACATTTAAAGTTGGTTTAAGTTGGCTTCAATTGATGGC AACTTAAGATAATCAGAATTTTCTTACCTTTTTAGCTTTACTGTTGAAAAGCCCTGTGATCTTGTACAAATCAT TTGCTTCTTGGGTAGTAATTTCTTTTACTCAAACGTGAGCTTTTGACTAGGTGAATGTAAATGTTCTTCTAGCTC TAATATCCTTTATTCTTTATATTTTCTAACAGATTCTGTGTAGTTGGATGAGCAGAGAACAAAAACAAAATAAT CCAGTGAGGAAAGCCCGTGAATAAACTTTCAGACCCGAGATCTACTCTCTAGCCTATTTTAAGCTCAACTTAA AAAGAAGAACTGTTCTCTGATTCTTTTCGCCTTCAATACTCAATGATTTAACTCCGCCCTCCTTCAAAAGAAAC AGCATTTCCTACTTTTATACTGTCTATATGATTGATTTGCACAACTCATCTGCCAGAAGAGCTGAGACATCCGT TCCCTTACAAGAAACTCTCCCCGGTAAGTAACCTCAGCTGCTTGGCCTATTAGTTAGCTTCTGAGATGAGTAA AAGCCTTAACAGGAAACCCATAGAAGACATTTGGCAAACACAAAGTGCTCATACAATTATCTTAAAATATAA TCTTTAAGATGAGGAAAGGGTCAGAGTTTAGAATGAGTTTCAGATTATTATAACATCAAAGATACAAAACATG ATTGTGAGTGAAAGACTTTAAAGGGAGCAATAGTATTTTAATAACTAACAGTCCTTACCTCTCAAAAGAAAGA TTTGCAGAGAGATGAGTCTTAGCTGAAATCTTGAAATCTTATCTTCTGCTAAGGAGAACTAAACCCTCTCCAG TGAAATGCCTTCTGAATATGTGCC

> NAME = 'nemestrina CCR5 mRNA.fa' : TYPE = DNA ATGGACTATCAAGTGTCAAGTCCAACCTATGACATCGATTATTATACATCGGAACCCTGCCAAAAAATCAATG TGAAACAAATCGCAGCCCGCCTCCTGCCTCCGCTCTACTCACTGGTGTTCATCTTTGGTTTTGTGGGCAACATA CTGGTCGTCCTCATCCTGATAAACTGCAAAAGGCTGAAAAGCATGACTGACATCTACCTGCTCAACCTGGCCA TCTCTGACCTGCTTTTCCTTCTTACTGTCCCCTTCTGGGCTCACTATGCTGCTGCCCAGTGGGACTTTGGAAATA CAATGTGTCAACTCTTGACAGGGCTCTATTTTATAGGCTTCTTCTCTGGAATCTTCTTCATCATCCTCCTGACAA TCGATAGGTACCTGGCTATCGTCCATGCTGTGTTTGCTTTAAAAGCCAGGACAGTCACCTTTGGGGTGGTGAC AAGTGTGATCACTTGGGTGGTGGCTGTGTTTGCCTCTCTCCCAGGAATCATCTTTACCAGATCTCAGAGAGAA GGTCTTCATTACACCTGCAGCTCTCATTTTCCATACAGTCAGTATCAATTCTGGAAGAATTTTCAGACATTAAA GATGGTCATCTTGGGGCTGGTCCTGCCGCTGCTTGTCATGGTCATCTGCTACTCGGGAATCCTGAAAACTCTGC TTCGGTGTCGAAACGAGAAGAAGAGGCACAGGGCTGTGAGGCTTATCTTCACCATCATGATTGTTTATTTTCT CTTCTGGGCTCCCTACAACATTGTCCTTCTCCTGAACACCTTCCAGGAATTCTTTGGCCTGAATAATTGCAGTA GCTCTAACAGGTTGGACCAAGCCATGCAGGTGACAGAGACTCTTGGGATGACACACTGCTGCATCAACCCCAT CATCTATGCCTTTGTCGGGGAGAAGTTCAGAAACTACCTCTTAGTCTTCTTCCAAAAGCACATTGCCAAACGCT TCTGCAAATGCTGTTCCATTTTCCAGCAAGAGGCTCCCGAGCGAGCAAGTTCAGTTTACACCCGATCCACTGG GGAGCAGGAAATATCTGTGGGCTTGTGA Anopheles CTLMA2: I believe this is straight genomic sequence as it had the following info with it: ref|NT_078265.1|:13663306-13664186 Anopheles gambiae str. PEST chromosome 2L (CDS 176..700)

GCCTTCTTCACGCATACCTCTTATCATCGCATCCCAATCGCAGTTGTACAGAAGGTGCTTCTACGATC ATTCAAGCTCACTCTCAGAAAACGTATAGTCTTTAGAAGTTTGTGTGGTTTTCTGTACTGTTATACTG TGTTCCTTTTTACAGAAAAAGTGGTAAACCATTTTTAAAATGAATTTAATGATCACAATTCTAATTGC TGTCATCACAGTGGTTCGTGGTGACCTATCGGTTCCTAAAACTGTGGATGGTAAGATTTTTGGGGTTT TTGTTATGGTCCAATTTAGGTTCGATTCACGTAATAATTTGTTATAACCATTTCCAGATATTCTTCAA CAAAACCCATGCCTTTGCCCATGCAAACCGTTCGAGGAGAAAGAATACTTCATTCCCATCAGTCGGG TAGAATAGAATTCCAAACCGTATCCAAATGCAACCTAAACCTTTATCACCCATTCCATCCTTCGCAG ACGAGCGACTGGTTCGGTGCCGTTGCCTACTGCCACAGCGTTGGCATGGAGCTGGCCGAGGTGCTGA ACGAAGACGAAGCACGAGCCATGGGCGAAGTGATCGCGGAGGAAGAATCCGACTCGGATGACGAG TTTTACTGGATCGGAGCGAATGATCTCGGCGTGCAGGGTACTTACCGGTGGGCATTAGCTGGACGGC CAGTGTTGTACAGCCAATGGGCTGCAGGTGAACCGAACCATGCTCGCGGTGAGAATGGCCAACAGC CCGCCGAACGATGCGTCGCTGTTGCGATGGACAAGTACGAGTGGAACGATTTCCAATGTACGCAGC AGAAGCCGTTCATCTGTCAACAGTTCCGAAAACATTGAAAAGTGTGAATAAATCGTGCGTTTTCTCG GCAGAAACA

Additional targets  Murine XSCID (Word from Andy is this doesn’t need additional site searches because of a natural target and other sites for Sce targeting).