Download What are CRISPRs

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
Transposons!
Jumping Genes
Barbara McClintock
Tandem Repeats
“Slippage”
CRISPRs
Clustered Short Regularly Spaced Palindromic Repeat
~ 35 to 37
nucleotides
Jump?
Slippage?
Exact count of
nuclotides
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
#
Nostoc PCC 7120
Nostoc punctiforme
Escherichia coli
28
16
13
9
4
4
GTTACTTACCATCACTTCCCCGCAAGGGGATGGAAAC
GTTTTAACTAACAAAAATCCCTATCAGGGATTGAAAC
GTTTCTATTAACACAAATCCCTATCAGGGATTGAAAC
GTTGCAACACCATATAATCCCTATTAGGGATTGAAAC
GTTAAAACCCTCTAAAATCCCTATCAGGGATTGAAAC
GTTGCAACCCTCCTTCCAGTAATGGGAGGGTTGAAAG
58
42
19
9
12
ATTGCAATTTCTCAAAATCCCTATTAGGGATTGAAAC
ATTGCAATTCATCAAAATCCCTATCAGGGATTGAAAC
ATTGCAATTCATCAAAATCCCTATTAGGGATTGAAAC
ATTGCAATTTATCAAAATCCCTATTAGGGATTGAAAC
GTGGCAACAACCCTCCAGGTACTGGGTGGGTTGAAAG
2-4
CGGTTTATCCCCG GCGGG-GAACAC
CTGGC
Similarity Between CRISPRs
Nostoc PCC 7120
28
16
13
9
4
4
GTTACTTACCATCACTTCCCCGCAAGGGGATGGAAAC
GTTTTAACTAACAAAAATCCCTATCAGGGATTGAAAC
GTTTCTATTAACACAAATCCCTATCAGGGATTGAAAC
GTTGCAACACCATATAATCCCTATTAGGGATTGAAAC
GTTAAAACCCTCTAAAATCCCTATCAGGGATTGAAAC
GTTGCAACCCTCCTTCCAGTAATGGGAGGGTTGAAAG
Nostoc punctiforme 58 ATTGCAATTTCTCAAAATCCCTATTAGGGATTGAAAC
42
19
9
12
Escherichia coli
2-4
ATTGCAATTCATCAAAATCCCTATCAGGGATTGAAAC
ATTGCAATTCATCAAAATCCCTATTAGGGATTGAAAC
ATTGCAATTTATCAAAATCCCTATTAGGGATTGAAAC
GTGGCAACAACCCTCCAGGTACTGGGTGGGTTGAAAG
CGGTTTATCCCCG
GCGGG—GAACAC
CTGGC
How did CRISPRS arise in
cyanobacteria genomes?
Anabaena
7120
Any matching CRISPR in Anabaena variabilis?
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
Copy #
28
Nostoc PCC 7120
16
13
9
4
4
GTTACTTACCATCACTTCCCCGCAAGGGGATGGAAAC
GTTTTAACTAACAAAAATCCCTATCAGGGATTGAAAC
GTTTCTATTAACACAAATCCCTATCAGGGATTGAAAC
GTTGCAACACCATATAATCCCTATTAGGGATTGAAAC
GTTAAAACCCTCTAAAATCCCTATCAGGGATTGAAAC
GTTGCAACCCTCCTTCCAGTAATGGGAGGGTTGAAAG
1
2
3
4
5
6
Blast
Anabaena
variabilis
?
How did CRISPRS arise in cyanobacteria genomes?
(SEQUENCE-SIMILAR-TO "CTTTCCGATCACATCACCCCGAAAGGGGATGGAAAC" IN avar)
 Nil
(SEQUENCE-SIMILAR-TO "**********************************************" IN avar)
 Nil
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
Copy
#
28
Nostoc PCC 7120 16
13
9
4
4
GTTACTTACCATCACTTCCCCGCAAGGGGATGGAAAC
GTTTTAATTCCTTTACCCCTCACGGGGATGGAAAC
GTTTCTATTAACACAAATCCCTATCAGGGATTGAAAC
GTTGCAACACCATATAATCCCTATTAGGGATTGAAAC
GTTAAAACCCTCTAAAATCCCTATCAGGGATTGAAAC
GTTGCAACCCTCCTTCCAGTAATGGGAGGGTTGAAAG
1
2
3
4
5
6
Blast
Anabaena
variabilis
?
Sequences Found In Cyanobacteria
Copy
#
28
Nostoc PCC 7120 16
13
9
4
4
GTTACTTACCATCACTTCCCCGCAAGGGGATGGAAAC
GTTTTAATTCCTTTACCCCTCACGGGGATGGAAAC
GTTTCTATTAACACAAATCCCTATCAGGGATTGAAAC
GTTGCAACACCATATAATCCCTATTAGGGATTGAAAC
GTTAAAACCCTCTAAAATCCCTATCAGGGATTGAAAC
GTTGCAACCCTCCTTCCAGTAATGGGAGGGTTGAAAG
0
2
147
35
146
0
0
0
0
0
0
0
1
2
3
4
5
6
Blast
Anabaena
variabilis
?
What conclusions can be drawn?
• Anabaena 7120 has at least four unique CRISPRs
that were found in Anabaena variabilis
Six matching CRISPRs inserted
common ancestor
of cyanobacteria
retained all six
CRISPRs
Anabaena 7120
lost two CRISPRs
Anabaena variabilis
What conclusions can be drawn?
• Anabaena 7120 has at least four unique CRISPRs
that were found in Anabaena variabilis
common ancestor
of cyanobacteria
six CRISPRs inserted
Anabaena 7120
four CRISPRs
inserted into A.var
Anabaena variabilis
The
The End
End
Special thanks to Dr. James Godde
• Assistant Professor at Monmouth, Illinois
• Contact number: 309-457-2350
• Email: [email protected]