Trinucleotide Repeat Diseases: CRISPR Cas9 PacBio no PCR Sequencing MFMER slide-1

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1 Trinucleotide Repeat Diseases: CRISPR Cas9 PacBio no PCR Sequencing 2015 MFMER slide-1

2 Fuch s Eye Disease TCF 4 gene Fuchs occurs in about 4% of the US population. Leads to deteriorating vision without corneal replacement surgery. Roughly 80% of Fuch s cases appear to be caused by expansion of a CAG repeat in a TCF-4 intron Transcription factor 4 (TCF-4) also known as immunoglobulin transcription factor 2 (ITF-2) is a protein that in humans is encoded by the TCF4 gene located on chromosome 18q21.2

3 TCF 4 sgrna design (BROAD) CGGAGCCCCCCCACCCATTTCTTTGCTGAACTTGCAATTCCGTGCGCCTCGGCGTGTTTCCCCCTCCCCCCTTCCCTCCGTCCCCT CCCCTCCCCGGAGAAGAGAGTTGGTGTTAAGAGTCAGGGATCTTGGCTGTGTGTCTGCGGATCTGTAGTGGCGGCGGCGGCGGCG GCGGCGGGGAGGCAGCAGGCGCGGGAGCGGGCGCAGGAGCAGGCGGCGGCGGTGGCGGCGGCGGTTAGACATGAACGCCGC CTCGGCGCCGGCGGTGCACGGAGAGCCCCTTCTCGCGCGCGGGCGGTAGGTACCGGCGCCTGCGGGGCTCGGCGGGGCGGAG GCGCCCGGCGGCGCGGGGTTCGGGCTCGGCGGCCCCGCACGCGGCTCCGCGCCTCCCGCGCCGCGGGCTCCCGGCGCCCGG CGCTCCCAGAAGAGACACCCCTTCCCCTCCCGCCGCTTCCCTCCCCCTCGCCGCCAGCCCCCCCGCCCCTCCCCTTGATGCCCCC TCGGAGGGACCGAGGACTTTGCCAGGGGCCTGACTTTAATTTTTATAACCCCTTTCTTTCACAAATTAGGGTGCTGGACAATTAGAGG ACCCGACCCTCCACTCCGCTCCCCCCAACCCTGTCACCCCACCCCTCCCCCTTTGGACGTGTTTTCACTCCAGAGACGCTCATCTT TAATTTCTTGATAGCTACTTTGAAAAGCGGAGGGGAGTGGTGTGTGCGAGTGTGTGTGCGTGTGCGAGTGTGAGTGTGCGAGGGGG GTGGGGTGCAGGGTGGGGGGAAAGTTGAGATCGGATCGTTCTCAGTAGTTTCTCTTTTCTCTGTGATCCATTCATTTCTCGCTCTACC TCCTGTTGCATAAAATGATGCGCTGAAGAGCGGCTTTTTTTTTTTCTTTCCCTGATTTGGTTTTATAGTGGATGTTACCAGTTTGATCGT CTCTTTGGAAATACAATATCGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCTATTGCTGCCTATTCCATCTTAAAAAAACAAAA CCAAACCCAACCCAACAACAAGTCTCCCATCCCTTCCTCCTTTCCACCTTAATCCCACCCGCCCCCACGTCCCCACCCCCAACCCC CCAGTCTCCAAAAATCCGATTGTGTTTTCTCCAAGGATTGGGACTGGATTTTCTGATTGCGGTTATTTCCATGTTTCTAGGTTTGTGTG ATTTTGCTAAAATGCATCACCAACAGCGAATGGCTGCCTTAGGGACGGACAAAGAGCTGAGTGATTTACTGGATTTCAGTGCGGTAA GAAAGAACGGTGGAAACTAACAACAGCTGTGAAAAAAACAAAACAAAAACCCAAACACTTCAGCTAGAAACCAGTAGGAATCTAAAG GACAGTAATAATTTTTAATTGGCTGAATCCTTGGTAAATATGAAGGTCTTTTTGACAAGTTTTTAACTATAATTTTGTGGTGTGATGGAAG ATTCAGGCTTTTTTTTTTTTTTGAGTTTTATTACTGGCCTTCAATTCCCTACCCACTGATTACCCCAAATAATGGAATCTCACCCCAGTG GAAAGCAAAAATAGACACCCCTAAAACTAAACCACCCCTAAAACTTGGCCATGTCTGAACACTGAGACTACTAATACTTTGCACACTA CTCTTCGTTTTATTTATTGTTTTTGGAAATGGAAAATAGAAAATAGGAGACCCAGTTGTCTCTTTAAAGTTTTAAGCTAATGATGCTTTGG ATTGGTAGGACCTGTTCCTTACATCTTACCTCCTAGTTACATCTTTTCCTAGGATTCTTAAAACTAGTATGGATATGCTGAGCATACATTC TTTAGAACCTTTTGGACTGTTTTGGTAAATTTCGTAGTCGTAGGATCAGCACAAAGCGGAACTTGACACACTTGTGGAGTTTTACGGC TGTACTTGGTCCTTCTCCATCCCTTTGCTTCCTTTTCCTAAACCAAGTCCCAGACATGTCAGGAGAATGAATTCATTTTTAATGCCAGA TGAGTTTGGTGTAAGATGCATTTGTAAAGCAAAATAAAAAGAATCCACAAAACACACAAATAAAATCCAAACCGCCTTCCAAGTGGGG CTCTTTCATGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTCC TCCTCCTCCTCCTCCTTCTCCTCCTCCTCCTCCTCTTCTAGACCTTCTTTTGGAGAAATGGCTTTCGGAAGTTTTGCCAGGAAACGTA GCCCTAGGCAGGCAGCTTTGCAGCCCCCTTTCTGCTTGTTGCACTTTCTCCATTCGTTCCTTTGCTTTTTGCAGGCTCTGACTCAGG GAAGGTGTGCATTATCCACTAGATACGTCGAAGAAGAGGGAAACCAATTAGGGTCGAAATAAATGCTGGAGAGAGAGGGAGTGAAAG

4 BROAD Institute TCF 4 cr/sgrna design CRISPR Enzyme - S. pyogenes Target Human Paste in 1500 bases from 5 end (repeat for 3 end) Quota 200 Submit Save (sgrna Picking results) excel

5 crrna or crrna-xt IDT Guide RNAs (grnas) contain the target-specific sequence for guiding Cas9 protein to a genomic location. Choose from crrnas or crrna XT, which form a grna duplex with tracrrna. Under challenging experimental conditions (e.g., high nuclease environments or with Cas9 mrna), use crrna XT or sgrna, which have additional chemical modifications for the highest level of stability and performance MFMER slide-5

6 Topo Cloning:TCF4 Gene Unique cutting site? NotI CGGAGCCCCCCCACCCATTTCTTTGCTGAACTTGCAATTCCGTGCGCCTCGGCGTGTTTCCCCCTCCCCCCTTC CCTCCGTCCCCTCCCCTCCCCGGAGAAGAGAGTTGGTGTTAAGAGTCAGGGATCTTGGCTGTGTGTCTGCGGAT CTGTAGTGGCGGCGGCGGCGGCGGCGGCGGGGAGGCAGCAGGCGCGGGAGCGGGCGCAGGAGCAGGCGGC GGCGGTGGCGGCGGCGGTTAGACATGAACGCCGCCTCGGCGCCGGCGGTGCACGGAGAGCCCCTTCTCGCG CGCGGGCGGTAGGTACCGGCGCCTGCGGGGCTCGGCGGGGCGGAGGCGCCCGGCGGCGCGGGGTTCGGGC TCGGCGGCCCCGCACGCGGCTCCGCGCCTCCCGCGCCGCGGGCTCCCGGCGCCCGGCGCTCCCAGAAGAGA CACCCCTTCCCCTCCCGCCGCTTCCCTCCCCCTCGCCGCCAGCCCCCCCGCCCCTCCCCTTGATGCCCCCTCG GAGGGACCGAGGACTTTGCCAGGGGCCTGACTTTAATTTTTATAACCCCTTTCTTTCACAAATTAGGGTGCTGGA CAATTAGAGGACCCGACCCTCCACTCCGCTCCCCCCAACCCTGTCACCCCACCCCTCCCCCTTTGGACGTGTTT TCACTCCAGAGACGCTCATCTTTAATTTCTTGATAGCTACTTTGAAAAGCGGAGGGGAGTGGTGTGTGCGAGTGT GTGTGCGTGTGCGAGTGTGAGTGTGCGAGGGGGGTGGGGTGCAGGGTGGGGGGAAAGTTGAGATCGGATCGT TCTCAGTAGTTTCTCTTTTCTCTGTGATCCATTCATTTCTCGCTCTACCTCCTGTTGCATAAAATGATGCGCTGAAG AGCGGCTTTTTTTTTTTCTTTCCCTGATTTGGTTTTATAGTGGATGTTACCAGTTTGATCGTCTCTTTGGAAATACAA TATCGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCTATTGCTGCCTATTCCATCTTAAAAAAACAAAACC AAACCCAACCCAACAACAAGTCTCCCATCCCTTCCTCCTTTCCACCTTAATCCCACCCGCCCCCACGTCCCCACC CCCAACCCCCCAGTCTCCAAAAATCCGATTGTGTTTTCTCCAAGGATTGGGACTGGATTTTCTGATTGCGGTTATT TCCATGTTTCTAGGTTTGTGTGATTTTGCTAAAATGCATCACCAACAGCGAATGGCTGCCTTAGGGACGGACAAA GAGCTGAGTGATTTACTGGATTTCAGTGCGGTAAGAAAGAACGGTGGAAACTAACAACAGCTGTGAAAAAAACAA AACAAAAACCCAAACACTTCAGCTAGAAACCAGTAGGAATCTAAAGGACAGTAATAATTTTTAATTGGCTGAATCC TTGGTAAATATGAAGGTCTTTTTGACAAGTTTTTAACTATAATTTTGTGGTGTGATGGAAGATTCAGGCTTTTTTTTT TTTTTGAGTTTTATTACTGGCCTTCAATTCCCTACCCACTGATTACCCCAAATAATGGAATCTCACCCCAGTGGAAA GCAAAAATAGACACCCCTAAAACTAAACCACCCCTAAAACTTGGCCATGTCTGAACACTGAGACTACTAATACTTT GCACACTACTCTTCGTTTTATTTATTGTTTTTGGAAATGGAAAATAGAAAATAGGAGACCCAGTTGTCTCTTTAAAG TTTTAAGCTAATGATGCTTTGGATTGGTAGGACCTGTTCCTTACATCTTACCTCCTAGTTACATCTTTTCCTAGGAT TCTTAAAACTAGTATGGATATGCTGAGCATACATTCTTTAGAACCTTTTGGACTGTTTTGGTAAATTTCGTAGTCGT AGGATCAGCACAAAGCGGAACTTGACACACTTGTGGAGTTTTACGGCTGTACTTGGTCCTTCTCCATCCCTTTGC TTCCTTTTCCTAAACCAAGTCCCAGACATGTCAGGAGAATGAATTCATTTTTAATGCCAGATGAGTTTGGTGTAAG ATGCATTTGTAAAGCAAAATAAAAAGAATCCACAAAACACACAAATAAAATCCAAACCGCCTTCCAAGTGGGGCTC TTTCATGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGC TGCTGCTCCTCCTCCTCCTCCTCCTTCTCCTCCTCCTCCTCCTCTTCTAGACCTTCTTTTGGAGAAATGGCTTTCG GAAGTTTTGCCAGGAAACGTAGCCCTAGGCAGGCAGCTTTGCAGCCCCCTTTCTGCTTGTTGCACTTTCTCCATT CGTTCCTTTGCTTTTTGCAGGCTCTGACTCAGGGAAGGTGTGCATTATCCACTAGATACGTCGAAGAAGAGGGAA ACCAATTAGGGTCGAAATAAATGCTGGAGAGAGAGGGAGTGAAAG

7 crrna Testing TCF4 Clone Mix crrna and tracrrna equal volume of 10uM. Incubate 50 degrees 5 min. slow cool to 25 degrees. Mix CRISPR buffer, Cas9, Guide RNA. Incubate 37 degrees 10 min. Add Heparin final conc. 3ug/ul. Incubate 37 degrees 3 min. Add 200 ng of cloned DNA incubate 37 degrees 1 hour. AMPure clean 0.45X AMPure. Fragment Analyzer or Gel

8 crrna Testing TCF4 Clone NEB Procedure Component Nuclease free water 10X Cas9 Nuclease Reaction Buffer 10uM crrna & 10uM tracrrna Pre Incubate 50 degrees 5 min slow cool to 25 1uM Cas ( Nuclease S. pyogenes) Reaction volume 30ul reaction 20ul 3ul 3ul Incubate 1.0ul 27ul 200 ng substrate DNA 3.0ul Total reaction volume 30.0ul Mix and pulse spin. Incubate 37 degrees 15 minutes Add 1ul Proteinase K (NEB #P8107S) Incubate Room temp. for 10 minutes. AATI Fragment Analysis

9 AATI Fragment Analyzer

10 AATI Fragment Analyzer

11 2015 MFMER slide-11 Workflow

12 2015 MFMER slide-12 TCF 4 Data Dr. Wieben- PacBio

13 Sample with no Repeat Expansion

14 Same sample analyzed with Brett Bowman s software from PacBio

15 Sample with Repeat Expansion note insertions in about half the reads

16 Same sample analyzed with Brett Bowman s software from PacBio

17 PacBio positive controls HTT

18 PacBio positive controls FMR-1

19 Hurdles of CRISPR Design and testing crrna Bioinformatics pipeline Labor Intensive Amount of upfront DNA (20ug) 2015 MFMER slide-19

20 Thank You! Ross Aleff 2015 MFMER slide-20