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Team:Arizona State/Project/CRISPR
From 2011.igem.org
CRISPR
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are a genomic feature of many prokaryotic and archeal species. CRISPR functions as an adaptive and inheritable immune system[38][50][31][36][40]. A CRISPR locus consists of a set of cas (CRISPR associated) genes, a leader, or promoter, sequence, and an array. This array consists of repeating elements along with "spacers". These spacer regions direct the CRISPR machinery to degrade or otherwise inactivate a complementary sequence in the cell.
Contents |
Engineered arrays
- By engineering a spacer complementary to T3 phage, increased survival was demonstrated[15][23][26][48][55].
- A customized spacer can prevent transformation of PC194 plasmids with a matching sequence[26].
CRISPR in E. coli
There are four crispr loci in E. coli. CRISPR1, the largest, is associated with eight cas genes[70]. In the classification scheme presented by Haft et al[13], these genes form the cse family, outlined below:
- casA, casB, casC, casD, casE, aka cse1, cse2, cse3, cse4, cas5e[13]:
CRISPR in P. furiosus
P. furiosus contains 7 crispr loci, along with 29 cas proteins in 2 gene clusters[33]. All 6 core cas proteins (cas1-cas6), as well as genes from the cmr, cst, and csa families are present.
Stages of the CRISPR pathway
There are 3 distinct stages of the CRISPR pathway: integration [15][51][20], expression, and adaptation. The cas subtype this section will refer to as an example is cse (E. coli).
Integration / Adaptation
In this step, DNA, commonly derived from phages and plasmids[45], is recognized and processed by cas proteins. Information from outside of the genome is recognized and incorporated into the leader end of an existing array. This involves cas1 and cas2[30][57]. The integration stage is the least understood aspect of the pathway.
Expression
In the expression stage, the CRISPR array is transcribed in its entirety, yielding pre-crRNA. This pre-crRNA is cleaved at repeat regions[70][7][25][29] by casE to yield crRNA. This crRNA is 61 bp long, consisting of a 31 bp spacer, flanked by repeat-derived segments on both ends[60] (8 bp at 5'[23][26][25], 21 bp forming a hairpin at 3', with a 5' hydroxyl group). crRNA is then bound to CASCADE, a protein complex consisting of casA, B, C, D, and E[60].
Interference
This stage requires cascade bound with crRNA, as well as cas3 [60]. The cascade complex may target DNA in the case of cse[23][26][51], or RNA in the cmr subtype[33]. Recognition of target DNA takes place by means of R loops[60][70][1]. An r loop is an RNA strand that has base paired with a complementary DNA strand, displacing the other identical DNA strand[1]. This base pairing between the crRNA spacer sequence and target strand may mark the region for interference by other proteins such as cas3[60].
CASCADE complex
This is a protein complex of casA-E, resembling a seahorse in shape[60]. Its full composition is 1x casA, 2x casB, 6x casC, 1x casD, 1x casE[60]. All protein components (casA-casE) are required for virus resistance[60]. This complex binds double stranded target DNA without need or enhancement by cofactors such as metal ions or ATP[60]. It also undergoes conformational changes when binding DNA[60].
Core cas genes
There are 6 “core” cas genes, found in a wide variety of organisms and here referred to as cas1-cas6[13].
cas1, cas2
cas1 is nearly universally conserved throughout organisms with CRISPR[30]. It is strongly implicated in the integration stage of the pathway[30][57]. cas1 is a metal-dependent (Mg, Mn) DNA specific endonuclease that generates an 80 bp fragment[30]. How is this converted into a ~32 bp spacer is unknown. cas2 is also involved in integration[30][57], and is a metal dependent endoribunuclease[22].
cas3
cas3 is not regulated by H-NS[39]. It cooperates with the cascade complex[23] in the interference stage. Cas3 has predicted ATP dependent helicase activity[4], as well as demonstrated ATP independent annealing of RNA to DNA[70]. It forms an r-loop with DNA, requiring magnesium or manganese as a co-factor[70], but has an antagonistic function in the presence of ATP, dissociating the r-loop.
The CRISPR array
Genetic information from previous encounters is stored in the array as spacers. These spacers are consistent in length (30-40 bp), and are flanked by repeating elements (also 30-40 bp). The repeating elements are usually partially palindromic, and form secondary structures when transcribed into pre-crRNA. These structures may be necessary for recognition and cleavage.
Prevention of self targeting (autoimmunity)
The 5' handle of crRNA allows self / nonself discrimination in the csm subtypetype[37]. In cse, regions flanking the proto spacer contain PAMs[37][12][20][28][19].
cas gene regulation
In E. coli (cse subtype), transcription of the cascade genes and CRISPR array is repressed by H-NS[45][41]. H-NS is a global repressor of transcription in many gram negative bacteria that binds AT rich sequences[14]. This repression is mediated by "DNA stiffening"[35], as well as formation of "DNA-protein-DNA" bridges[10]. The creation of an H-NS knockout can be shown to increase expression of cas genes[45][5]. This correlates with phage sensitivity[45]. Transcription is antagonistically[24] de-repressed by LeuO[45], a protein of the lysR transcription factor family[24] near the leuABCD (leucine synthesis[2]) operon[11]. LeuO expression is also repressed by H-NS[3][6]. Expression of H-NS repressed proteins can be manipulated by plasmid-encoded leuO in a consitutive promoter[32]. Plasmids: pCA24N (lac1 promoter), pKEDR13 (pTac promoter), pNH41 (IPTG). Increased LeuO expression leads to increased expression of casABCDE, cas1, and cas2[45][32], but does not affect cas3 expression[45]. Constitutively expressing leuO had a stronger affect than knocking out H-NS[45].
Classfication of crispr systems
- Haft (2005) [13]: Recognition of core cas genes (1-6). Organized remaining genes into 9 subtypes: Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, Mtube, RAMP.
- Makarova (2011) [72]: Classification into I, II, and III subtypes, based on mechanism of action as well as homology. These subtypes correspond with the 9 given by Haft to a large extent:
- I-A: Apern
- I-B: Tneap / Hmari
- I-C: Dvulg
- I-D
- I-E: Ecoli
- I-F: Ypest
- II-A: Nmeni
- II-B: Nmeni
- III-A: Mtube
- III-B: Polymerase-RAMP
- Comprehensive listing of cas genes:
| Name [65] | Name [15] | Name[25] | I-A | I-B | I-C | I-D | I-E | I-F | II-A | II-B | III-A | III-B | I-U | III-U | U | COG | TIGR | Ramp [65] | cascade-like complex [65] | putative function | grouping [75] |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| cas_HTH | |||||||||||||||||||||
| cas1 | cas1 | cas1 | y | y | y | y | y | y | y | y | y | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG1518 1518] | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR00287 00287] | Nuclease [65] | ||||||
| cas10 | cmr2 | y | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG1353 1353] | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR02577 02577] | y | Polymerase [65] | ||||||||||||||
| csm1 | y | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR02578 02578] | y | |||||||||||||||||
| csx11 | y | y | y | ||||||||||||||||||
| cas10d | csc3 | y | y | ||||||||||||||||||
| cas2 | cas2 | cas2 | y | y | y | y | y | y | y | y | y | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG1343 1343], [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG3512 3512] | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01573 01573] | |||||||
| cas3' | cas3 | cas3 | y | y | y | y | y | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG1203 1203] | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01587 01587] | Helicase, nuclease [15] | ||||||||||
| cas3 | y | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG2254 2254] | ||||||||||||||||||
| cas4 | cas4 | y | y | y | y | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG1468 1468] | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR00372 00372] | RecB exonuclease [65][8] | ||||||||||||
| csa1 | y | y | y | y | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01896 01896] | |||||||||||||||
| cas5 | cas5d | casD | y | y | y | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG1688 1688] | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01876 01876] | y | y | N-terminal domain [15], crRNA processing [65] | cas5 | |||||||||
| cas5e | y | y | y | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01868 01868] | y | y | cas5 | |||||||||||||
| cas5h | y | y | y | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR02592 02592] | y | y | cas5 | |||||||||||||
| cas5t | y | y | y | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01895 01895] | y | y | cas5 | |||||||||||||
| cmx5 | y | y | y | y | y | y | cas5 | ||||||||||||||
| csa5a | y | y | y | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01874 01874] | y | y | cas5 | |||||||||||||
| cas6 | cas6 | y | y | y | y | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG1583 1583], [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG5551 5551] | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01877 01877] | y | crRNA processing [65] | cas6 | ||||||||||
| cmx6 | y | y | y | y | y | y | cas6 | ||||||||||||||
| cas6e | cse3 | casE | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01907 01907] | y | cas6 | |||||||||||||||
| cas6f | csy4 | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR02563 02563] | y | cas6 | ||||||||||||||||
| cas7 | csa2 | casC | y | y | y | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG1857 1857], [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG3649 3649] | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR02583 02583] | y | y | Regulation [15] | cas7 | |||||||||
| csd2 | y | y | y | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR02589 02589] | y | y | cas7 | |||||||||||||
| cse4 | y | y | y | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01869 01869] | y | y | cas7 | |||||||||||||
| csh2 | y | y | y | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR02590 02590] | y | y | cas7 | |||||||||||||
| cst2 | y | y | y | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR02585 02585] | y | y | cas7 | |||||||||||||
| cas8a1 | CXXC-CXXC | y | y | ||||||||||||||||||
| cmx1 | y | y | |||||||||||||||||||
| cst1 | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01908 01908] | y | ||||||||||||||||||
| csx13 | y | y | |||||||||||||||||||
| csx8 | y | y | |||||||||||||||||||
| cas8a2 | csa4 | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01914 01914] | y | |||||||||||||||||
| csx9 | y | y | |||||||||||||||||||
| cas8b | TM1802 | y | y | ||||||||||||||||||
| csh1 | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR02591 02591] | y | ||||||||||||||||||
| cas8c | csd1 | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01863 01863] | y | |||||||||||||||||
| csp2 | y | y | |||||||||||||||||||
| cas9 | csn1 | y | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG3513 3513] | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01865 01865] | HNH endonuclease [15] | |||||||||||||||
| csx12 | y | y | |||||||||||||||||||
| cmr1 | cmr1 | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG1367 1367] | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01894 01894] | y | y | cas7 | ||||||||||||||
| cmr3 | cmr3 | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG1769 1769] | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01888 01888] | y | y | cas5 | ||||||||||||||
| cmr4 | cmr4 | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG1336 1336] | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR02580 02580] | y | y | cas7 | ||||||||||||||
| cmr5 | cmr5 | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG3337 3337] | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01881 01881] | y | ||||||||||||||||
| cmr6 | cmr6 | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG1604 1604] | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01898 01898] | y | y | cas7 | ||||||||||||||
| csa5 | csa5 | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01878 01878] | y | |||||||||||||||||
| csaX | csaX | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR03876 03876] | ||||||||||||||||||
| csb1 | GSU0053 | y | y | ||||||||||||||||||
| csb2 | y | y | |||||||||||||||||||
| csb3 | y | y | |||||||||||||||||||
| csc1 | csc1 | y | y | cas5 | |||||||||||||||||
| csc2 | csc2 | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG1337 1337] | y | y | cas7 | ||||||||||||||||
| cse1 | cse1 | casA | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR02547 02547] | y | ||||||||||||||||
| cse2 | cse2 | casB | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR02548 02548] | y | ||||||||||||||||
| csf1 | csf1 | y | |||||||||||||||||||
| csf2 | csf2 | y | y | cas7 | |||||||||||||||||
| csf3 | csf3 | y | y | ||||||||||||||||||
| csf4 | csf4 | y | |||||||||||||||||||
| csm2 | csm2 | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01870 01870] | y | |||||||||||||||||
| csm3 | csc2 | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG1337 1337] | y | y | cas7 | |||||||||||||||
| csm3 | y | y | y | cas7 | |||||||||||||||||
| csm4 | csm4 | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG1567 1567] | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01903 01903] | y | y | cas5 | ||||||||||||||
| csm5 | csm5 | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG1332 1332] | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01899 01899] | y | y | cas7 | ||||||||||||||
| csm6 | APE2256 | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG1517 1517] | transcription regulation [65] | |||||||||||||||||
| csm6 | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR02672 02672] | |||||||||||||||||||
| csn2 | csn2 | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01866 01866] | ||||||||||||||||||
| csx1 | DXTHG | y | [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG1517 1517], [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=COG4006 4006] | Helix-turn-helix, transcription regulation [15] | |||||||||||||||||
| NE0113 | y | ||||||||||||||||||||
| TIGR02710 | y | ||||||||||||||||||||
| csa3 | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR01884 01884] | |||||||||||||||||||
| csx1 | y | ||||||||||||||||||||
| csx2 | y | ||||||||||||||||||||
| csx10 | csx10 | y | y | ||||||||||||||||||
| csx14 | y | ||||||||||||||||||||
| csx15 | |||||||||||||||||||||
| csx16 | VVA1548 | y | |||||||||||||||||||
| csx17 | y | ||||||||||||||||||||
| csx3 | csx3 | y | |||||||||||||||||||
| csy1 | csy1 | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR02564 02564] | y | |||||||||||||||||
| csy2 | csy2 | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR02565 02565] | y | y | cas5 | |||||||||||||||
| csy3 | csy3 | y | [http://www.ebi.ac.uk/interpro/ISignature?ac=TIGR02566 02566] | y | cas7 |
