Cyanobacteria’s Repeat Sequences… where did they come from? Molly Sergio

Overview of Presentation unique DNA repeats Cyanobacteria genomes Similarities? Research !

Eureaka! Repeated Sequences! Dispersed Repeats Tandem Repeats CRISPRs

Dispersed Repeats Jumping Genes Transposons! Barbara McClintock

Tandem Repeats “Slippage”

CRISPRs Jump? Slippage? Where do CRISPRs come from? Clustered Short Regularly Spaced Palindromic Repeat ~ 35 to 37 nucleotides Jump? Exact count of nuclotides Slippage? Non-CRISPR sequence would be duplicated Where do CRISPRs come from? Picture credit: James Godde

Example of CRISPRs in Npun

CRISPR Sequences Found In Cyanobacteria Copy # 28 GTTACTTACCATCACTTCCCCGCAAGGGGATGGAAAC 16 GTTTTAACTAACAAAAATCCCTATCAGGGATTGAAAC 13 GTTTCTATTAACACAAATCCCTATCAGGGATTGAAAC 9 GTTGCAACACCATATAATCCCTATTAGGGATTGAAAC 4 GTTAAAACCCTCTAAAATCCCTATCAGGGATTGAAAC 4 GTTGCAACCCTCCTTCCAGTAATGGGAGGGTTGAAAG Nostoc PCC 7120 58 ATTGCAATTTCTCAAAATCCCTATTAGGGATTGAAAC 42 ATTGCAATTCATCAAAATCCCTATCAGGGATTGAAAC 19 ATTGCAATTCATCAAAATCCCTATTAGGGATTGAAAC 9 ATTGCAATTTATCAAAATCCCTATTAGGGATTGAAAC 12 GTGGCAACAACCCTCCAGGTACTGGGTGGGTTGAAAG Nostoc punctiforme 2-4 CGGTTTATCCCCG GCGGG-GAACAC CTGGC Escherichia coli

Similarity Between CRISPRs 28 GTTACTTACCATCACTTCCCCGCAAGGGGATGGAAAC 16 GTTTTAACTAACAAAAATCCCTATCAGGGATTGAAAC 13 GTTTCTATTAACACAAATCCCTATCAGGGATTGAAAC 9 GTTGCAACACCATATAATCCCTATTAGGGATTGAAAC 4 GTTAAAACCCTCTAAAATCCCTATCAGGGATTGAAAC 4 GTTGCAACCCTCCTTCCAGTAATGGGAGGGTTGAAAG Nostoc PCC 7120 58 ATTGCAATTTCTCAAAATCCCTATTAGGGATTGAAAC 42 ATTGCAATTCATCAAAATCCCTATCAGGGATTGAAAC 19 ATTGCAATTCATCAAAATCCCTATTAGGGATTGAAAC 9 ATTGCAATTTATCAAAATCCCTATTAGGGATTGAAAC 12 GTGGCAACAACCCTCCAGGTACTGGGTGGGTTGAAAG Nostoc punctiforme CGGTTTATCCCCG GCGGG—GAACAC CTGGC Escherichia coli 2-4

How did CRISPRS arise in cyanobacteria genomes? Anabaena 7120 Any matching CRISPR in Anabaena variabilis? compare Any non-CRISPR sequences match? Anabaena variabilis ? ?

Locating a CRISPR in a7120 1 36 (SEQUENCE-SIMILAR-TO "CTTTCCGATCACATCACCCCGAAAGGGGATGGAAAC" IN a7120) Query Q-start Q-end Subject S-start S-end ID value 1. "Seq1" 1 36 #$A7120.CHROMOSOME 5647145 5647110 100% 2. "Seq1" 1 36 #$A7120.CHROMOSOME 5647076 5647041 100% 3. "Seq1" 1 36 #$A7120.CHROMOSOME 5646995 5646960 100% 4. "Seq1" 1 36 #$A7120.CHROMOSOME 5646922 5646887 100% 5. "Seq1" 1 36 #$A7120.CHROMOSOME 5646849 5646814 100% 6. "Seq1" 1 36 #$A7120.CHROMOSOME 5646774 5646739 100% 7. "Seq1" 1 36 #$A7120.CHROMOSOME 5646703 5646668 100% 8. "Seq1" 1 36 #$A7120.CHROMOSOME 5646635 5646600 100% 9. "Seq1" 1 36 #$A7120.CHROMOSOME 5646562 5646527 100% 10."Seq1" 1 36 #$A7120.CHROMOSOME 5646490 5646455 100%

Locating in between an a7120 CRISPR (FOR-EACH coord-pair IN * AS left-coord = (+ (FIRST coord-pair) 1) AS right-coord = (- (SECOND coord-pair) 1) AS seq = (SEQUENCE-OF A7120.chromosome FROM left-coord TO right-coord) COLLECT seq) Non-coding sequences (in between CRISPRs) 1-2. "GAGAGACTGGAAACAATTTCTATAGCGATGTCGGAT" 2-3. "TCAATCATCTTTGCATTATATCCTGAAATTACAAGAT" 3-4. "CTCCCTTAGCCACTCTAGGATTTGTGACTGTT" 4-5. "ATGATTATCGAGGAATCTCGAAAATAGGACGTCGA" 5-6. "AAGCGACCATCGCTTTTTGCACGAACAGCAGATGGAACG" 6-7. "TCCTGCTCCAAGCATTAGTCCTTCGGAGATTAAAAAC" 7-8. "TAACTACTGCAAGCGTTGTGCAAGCAGCAATACCTGC" 8-9. "TGCACATTGATATAAACGAAGCTAAAAAAGCCTCTACCAATATAA" 9-10."TCTTGCATACAAAGCTGCATTTCTAGATGACAA"

How did CRISPRS arise in cyanobacteria genomes? GAGAGACTGGAAACAATTTCTATAGCGATGTCGGAT Anabaena 7120 CTTTCCGATCACATCACCCCGAAAGGGGATGGAAAC Compare CRISPR similarity Compare similarity Anabaena variabilis

? CRISPR Sequences Found In Cyanobacteria Anabaena variabilis Blast Copy # 28 GTTACTTACCATCACTTCCCCGCAAGGGGATGGAAAC 16 GTTTTAACTAACAAAAATCCCTATCAGGGATTGAAAC 13 GTTTCTATTAACACAAATCCCTATCAGGGATTGAAAC 9 GTTGCAACACCATATAATCCCTATTAGGGATTGAAAC 4 GTTAAAACCCTCTAAAATCCCTATCAGGGATTGAAAC 4 GTTGCAACCCTCCTTCCAGTAATGGGAGGGTTGAAAG Nostoc PCC 7120 1 2 3 4 5 6 Anabaena variabilis Blast ?

How did CRISPRS arise in cyanobacteria genomes? (SEQUENCE-SIMILAR-TO "CTTTCCGATCACATCACCCCGAAAGGGGATGGAAAC" IN avar) Nil (SEQUENCE-SIMILAR-TO "**********************************************" IN avar) Use a different CRISPR and its non-CRISPR sequences from a7120… (SEQUENCE-SIMILAR-TO "GTTTTAATTCCTTTACCCCTCACGGGGATGGAAAC" IN avar) Query Q-start Q-end Subject S-start S-end E-value 1. "Seq1" 1 35 #$A29413.Contig256 360180 360074 1.0d-13 2. "Seq1" 1 35 #$A29413.Contig256 360039 360005 1.0d-13 (SEQUENCE-SIMILAR-TO "**********************************************" IN avar) Nil

? Sequences Found In Cyanobacteria Anabaena variabilis Blast Copy # 28 GTTACTTACCATCACTTCCCCGCAAGGGGATGGAAAC 16 GTTTTAATTCCTTTACCCCTCACGGGGATGGAAAC 13 GTTTCTATTAACACAAATCCCTATCAGGGATTGAAAC 9 GTTGCAACACCATATAATCCCTATTAGGGATTGAAAC 4 GTTAAAACCCTCTAAAATCCCTATCAGGGATTGAAAC 4 GTTGCAACCCTCCTTCCAGTAATGGGAGGGTTGAAAG Nostoc PCC 7120 1 2 3 4 5 6 Anabaena variabilis Blast ?

? Sequences Found In Cyanobacteria Anabaena variabilis Blast Copy # 28 GTTACTTACCATCACTTCCCCGCAAGGGGATGGAAAC 16 GTTTTAATTCCTTTACCCCTCACGGGGATGGAAAC 13 GTTTCTATTAACACAAATCCCTATCAGGGATTGAAAC 9 GTTGCAACACCATATAATCCCTATTAGGGATTGAAAC 4 GTTAAAACCCTCTAAAATCCCTATCAGGGATTGAAAC 4 GTTGCAACCCTCCTTCCAGTAATGGGAGGGTTGAAAG 2 147 35 146 Nostoc PCC 7120 1 2 3 4 5 6 Anabaena variabilis Blast ?

What conclusions can be drawn? Anabaena 7120 has at least four unique CRISPRs that were found in Anabaena variabilis common ancestor of cyanobacteria Six matching CRISPRs inserted retained all six CRISPRs lost two CRISPRs Anabaena 7120 Anabaena variabilis

What conclusions can be drawn? Anabaena 7120 has at least four unique CRISPRs that were found in Anabaena variabilis Anabaena variabilis common ancestor of cyanobacteria Anabaena 7120 six CRISPRs inserted four CRISPRs inserted into A.var

The End The End Special thanks to Dr. James Godde Assistant Professor at Monmouth, Illinois Contact number: 309-457-2350 Email: jgodde@monm.edu