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

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

3. Eureaka! Repeated Sequences!
Dispersed Repeats
Tandem Repeats
CRISPRs

4. Dispersed Repeats
Jumping Genes
Transposons!
Barbara McClintock

5. Tandem Repeats
“Slippage”

6. 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

7. Example of CRISPRs in Npun

8. 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
CGGTTTATCCCCG GCGGG-GAACAC
CTGGC
Escherichia coli

9. 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

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

11. Locating a CRISPR in a7120
(SEQUENCE-SIMILAR-TO "CTTTCCGATCACATCACCCCGAAAGGGGATGGAAAC" IN a7120)
Query Q-start Q-end Subject S-start S-end ID value
1. "Seq1" #$A7120.CHROMOSOME %
2. "Seq1" #$A7120.CHROMOSOME %
3. "Seq1" #$A7120.CHROMOSOME %
4. "Seq1" #$A7120.CHROMOSOME %
5. "Seq1" #$A7120.CHROMOSOME %
6. "Seq1" #$A7120.CHROMOSOME %
7. "Seq1" #$A7120.CHROMOSOME %
8. "Seq1" #$A7120.CHROMOSOME %
9. "Seq1" #$A7120.CHROMOSOME %
10."Seq1" #$A7120.CHROMOSOME %

12. 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"

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

14. ? 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 ?

15. 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" #$A29413.Contig d-13
2. "Seq1" #$A29413.Contig d-13
(SEQUENCE-SIMILAR-TO "**********************************************" IN avar)
Nil

16. ? 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 ?

17. ? 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 ?

18. 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

19. 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

20. The End The End Special thanks to Dr. James Godde
Assistant Professor at Monmouth, Illinois
Contact number: