CRISPR-associated Proteins Sarah Pyfrom sapyfrom@davidson.edu

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?

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

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

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)

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

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

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

Often found with Cas1, and Cas6. Share and N-terminal region of about 43 amino acids in length Are usually 210-265 amino acids long Characterized by GhGxxxxxGhG motif, where h indicates a hydrophobic residue, at the C-terminus From: EMBL IPR013422 profile page (: http://www.ebi.ac.uk/interpro/IEntry?ac=IPR013422) From: Sanger PF09559 Profile page ( http://pfam.sanger.ac.uk/family/PF09559)

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.

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

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: http://www.youtube.com/watch?v=D- 77BvIOLd0

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

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

Phylogenetic tree comparing amino acid sequences for all CAS-proteins 2 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

Cas 1 and Cas2 did not change

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

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

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

…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.

Questions?

References Kunin, V., Sorek, R., Hugenholtz, P. (2007) Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biology.http://genomebiology.com/2007/8/4/R61. 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. http://www.ncbi.nlm.nih.gov/pubmed/16292354. Accessed 24 Nov 2009.