1. CRISPR-associated Proteins
Sarah Pyfrom

2. Research Questions
What Cas-proteins does our species share with the 10 other species we chose to study?
If so, how do they compare?
How do Cas-proteins function in relation to CRISPR units?
[Edit]:
Why did JGI change its annotation?

3. Cas Proteins
Proteins that are almost always associated with (near) CRISPR sequences
Originally four major families
Now, at least 45 families total

4. JGI annotation Cas1 Cas2 Cas3 Cas4 TM1800 TM1801 Cas1 Cas2 Cas4 Cas5
“Old” Cas-Proteins
“New” Cas-Proteins
Cas1
Cas2
Cas3
Cas4
TM1800
TM1801
Cas1
Cas2
Cas4
Cas5
Cas6
Csh1
Csh2

5. Changes: TM1800= Cas5 TM1801=Csh2 Hypothetical protein = Csh1
Part of hypothetical protein = Cas6
Cas3 = hypothetical protein
Cas4:
MTDSSGDPVDRFLAAARDESAELPFRLTGVMFQYYVVCER ELWFLSRDVEIDRDTPAIVRGSDVDDSAYADKRRDVRVDGII AIDVLDSGEILEVKPSSSMTEPARLQLLFYLWYLDRVTGVEK TGVLAHPAEKRRETVELTPETSAEVESAIEGIRAVVTAESPPP AEEKPVCDSCAYHDFCWSC (red = original Cas4)

6. Map of CRISPR region TM1800 TM1801 Transposases Cas3
Hypothetical proteins
CRISPR
Cas1
Unidentified
Csh1
Cas2
Cas4
Cas5
Cas6
Csh2

7. Cas1 (from Sulfolobus solfataricus)
high-affinity nucleic acid binding protein
binds DNA, RNA and DNA–RNA hybrid
sequence non-specific in a multi-site binding mode
promotes the hybridization of complementary nucleic acid strands.
From: SSO1450 – A CAS1 protein from Sulfolobus solfataricus P2 with high affinity for RNA and DNA

8. Usually similar to helicases
Unwinds double-stranded DNA
Thought to be involved in DNA metabolism and repair
Often resemble Rec-B exonucleases
Break down nucleic acid strains
Thought to be involved in DNA metabolism
Cas2 function unknown
From Genbank

9. Often found with Cas1, and Cas6.
Share and N-terminal region of about 43 amino acids in length
Are usually amino acids long
Characterized by GhGxxxxxGhG motif, where h indicates a hydrophobic residue, at the C-terminus
From: EMBL IPR profile page (:
From: Sanger PF09559 Profile page (

10. Csh1 and Csh2? Protein families determined for ease of alignment
Often large differences between species
Alignment easier if protein “soup” is divided into more readily- compared subgroups.

11. CRISPRs thought to create stable secondary RNA structures
Spacers remain associated with their DR neighbors.
Provide a way for Cas- Proteins to recognize the spacers and facilitate immune response.
From: Evolutionary conservation of sequence and secondary structures in CRISPR repeats

12. Cas-Proteins and Immunity
Thought to act like Slicer and Dicer (eukaryotic counterpart)
Create siRNA that will inhibit/break down invading RNA
Not known if Cas-proteins are involved in integrating pathogenic DNA into spacers
Video of eukaryotic siRNA process: 77BvIOLd0

13. Alignments of Cas
Compared Cas1, Cas2, Cas3 etc. proteins across all 10 species…

14. Comparison with other species: (based on “old” proteins)
Cas1
Cas2
Cas3
Cas4
TM1800
TM1801
H: vallismortis
H. volcanii
H. sulfurifontis X
H. sinaiiensis
H. californiae
H. utahensis
H. mucosum
H. mediteranei
H. denitrificans
H. mukohataei

15. Phylogenetic tree comparing amino acid sequences for all CAS-proteins2 2
Halomicrobium mukohataei 2
Haloarcula sinaiiensis 2
1800
Haloarcula californiae 1 4
Haloferax dentrificans 3 1
Haloferax mediteranei
1801
Haloferax sulfurifontis
1801
1801
Haloferax mucosum
1801
1801
Halorhabdus utahensis 3 1 1
1800 2 3 3 2
1800 1
1800
1800 1 3
1801 4 4 4 4 4 1 4
1800 3 3
1800
1801

16. Cas 1 and Cas2 did not change

17. Cas 4 JGI revision shortened this protein
Would expect low sequence similarity near end of protein

18. TM1801 (Csh2) Revision by JGI simply renamed this protein
Would expect sequence similarity

19. Map of CRISPR region TM1800 TM1801 Transposases Cas3
Hypothetical proteins
CRISPR
Cas1
Unidentified
Csh1
Cas2
Cas4
Cas5
Cas6
Csh2

20. …but we do know everything that everybody else knows.
In conclusion:
We don’t know much….
…but we do know everything that everybody else knows.

21. Questions?

22. References
Kunin, V., Sorek, R., Hugenholtz, P. (2007) Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biology. Accessed 24 Nov, 2009.
Haft, D.H., Selengut, J. Mongodin, EF., Nelson, K.E. (2005). A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput Biol. Accessed 24 Nov 2009.