Team:Arizona State/Project/CRISPR

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{{:Team:Arizona State/Templates/main|title=CRISPR|content=
{{:Team:Arizona State/Templates/main|title=CRISPR|content=
 
 
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''See [[Team:Arizona State/Glossary|glossary]] for explanation of various abbreviations used on this page.''
<p>'''C'''lustered '''R'''egularly '''I'''nterspaced '''S'''hort '''P'''alindromic '''R'''epeats (CRISPR) are a genomic feature of many prokaryotic and archeal species. CRISPR functions as an adaptive and inheritable immune system{{:Team:Arizona State/Templates/ref|38}}{{:Team:Arizona State/Templates/ref|50}}{{:Team:Arizona State/Templates/ref|31}}{{:Team:Arizona State/Templates/ref|36}}{{:Team:Arizona State/Templates/ref|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.</p>
<p>'''C'''lustered '''R'''egularly '''I'''nterspaced '''S'''hort '''P'''alindromic '''R'''epeats (CRISPR) are a genomic feature of many prokaryotic and archeal species. CRISPR functions as an adaptive and inheritable immune system{{:Team:Arizona State/Templates/ref|38}}{{:Team:Arizona State/Templates/ref|50}}{{:Team:Arizona State/Templates/ref|31}}{{:Team:Arizona State/Templates/ref|36}}{{:Team:Arizona State/Templates/ref|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.</p>
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== CRISPR in ''Pyrococcus  furiosus''==
== CRISPR in ''Pyrococcus  furiosus''==
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<p>''P. furiosus'' contains 7 CRISPR loci, along with 29 Cas genes in 2 gene clusters{{:Team:Arizona State/Templates/ref|33}}. All 6 core Cas genes (cas1-cas6), as well as genes from the Cmr, Cst, and Csa families are present. Cmr1-6 have been found to form a Cascade-like complex that targets RNA in in-vitro experiments{{:Team:Arizona State/Templates/ref|33}}.</p>
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<p>''P. furiosus'' contains 7 CRISPR loci, along with 29 Cas genes in 2 gene clusters{{:Team:Arizona State/Templates/ref|33}}. All 6 core Cas genes (cas1-cas6), as well as genes from the Cmr (type III), Cst (type I), and Csa (type I) families are present. Cmr1-6 have been found to form a Cascade-like complex that targets RNA in in-vitro experiments{{:Team:Arizona State/Templates/ref|33}}.</p>
[[Image:Arizona State P furiosus CRISPR]]
[[Image:Arizona State P furiosus CRISPR]]
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== Core Cas genes ==
== Core Cas genes ==
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<p>There are 6 “core” Cas genes, found in a wide variety of organisms and here referred to as cas1-cas6{{:Team:Arizona State/Templates/ref|13}}.</p>
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<p>There are 6 “core” Cas genes, found in a wide variety of organisms and here referred to as Cas1-Cas6{{:Team:Arizona State/Templates/ref|13}}.</p>
=== Cas1, Cas2 ===
=== Cas1, Cas2 ===
<p>Cas1 is nearly universally conserved throughout organisms with CRISPR{{:Team:Arizona State/Templates/ref|30}}. It is strongly implicated in the integration stage of the pathway{{:Team:Arizona State/Templates/ref|30}}{{:Team:Arizona State/Templates/ref|57}}. ''cas1'' is a metal-dependent (Mg, Mn) DNA-specific endonuclease that generates an 80 bp fragment{{:Team:Arizona State/Templates/ref|30}}. How this is converted into an ~32 bp spacer is unknown.</p>
<p>Cas1 is nearly universally conserved throughout organisms with CRISPR{{:Team:Arizona State/Templates/ref|30}}. It is strongly implicated in the integration stage of the pathway{{:Team:Arizona State/Templates/ref|30}}{{:Team:Arizona State/Templates/ref|57}}. ''cas1'' is a metal-dependent (Mg, Mn) DNA-specific endonuclease that generates an 80 bp fragment{{:Team:Arizona State/Templates/ref|30}}. How this is converted into an ~32 bp spacer is unknown.</p>
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<p>Cas2 is also involved in integration{{:Team:Arizona State/Templates/ref|30}}{{:Team:Arizona State/Templates/ref|57}}, and is a metal dependent endoribunuclease{{:Team:Arizona State/Templates/ref|22}}.</p>
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<p>Cas2 is also involved in integration{{:Team:Arizona State/Templates/ref|30}}{{:Team:Arizona State/Templates/ref|57}}, and is a metal dependent endoribonuclease{{:Team:Arizona State/Templates/ref|22}}.</p>
=== Cas3 ===
=== Cas3 ===

Revision as of 00:43, 26 September 2011


CRISPR


ASU Logo.png
 

See glossary for explanation of various abbreviations used on this page.

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

E. coli contains a type I CRISPR system. There are four CRISPR loci in Escherichia coli K-12 substr. MG1655. 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: casA, casB, casC, casD, casE, aka cse1, cse2, cse3, cse4, cas5e[13]. These 5 proteins combine to form the Cascade complex[60]. This is a protein complex of all 5 Cse genes, resembling a seahorse in shape[60]. Its full composition is 1x casA, 2x casB, 6x casC, 1x casD, 1x casE[60]. Specifically, casE cleaves pre-crRNA[23], and casA and casB can be omitted without affecting crRNA generation, but are necessary for phage 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][76].

cas gene transcription is repressed by H-NS[67], and de-repressed by leuO[45] or baeR[54]

.

caption=Structure of the CRISPRI locus in E. coli. 3 promoters have been identified[75]: Pcrispr1, Pcas, and anti-Pcas.

CRISPR in Pyrococcus furiosus

P. furiosus contains 7 CRISPR loci, along with 29 Cas genes in 2 gene clusters[33]. All 6 core Cas genes (cas1-cas6), as well as genes from the Cmr (type III), Cst (type I), and Csa (type I) families are present. Cmr1-6 have been found to form a Cascade-like complex that targets RNA in in-vitro experiments[33].

File:Arizona State P furiosus CRISPR

CRISPR in Bacillus halodurans C-125

B. halodurans contains 6 Cmr genes (Cmr1-6) in a single locus. This is a type III CRISPR system.

File:Arizona State B halodurans CRISPR

CRISPR in Listeria innocua

L. innocua contains a type II CRISPR system. A single gene (Cas9) has been shown to be necessary for the expression and inactivation stages of the pathway[62]. A separate trans-encoded small RNA (tracrRNA) binds with the repeat segment of the pre-crRNA[59], followed by cleavage by RNase III and binding with Cas9.

File:Arizona State L innocua CRISPR

Stages of the CRISPR pathway

There are 3 distinct stages of the CRISPR pathway: integration[15][51][20], expression, and adaptation.

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 currently 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] to yield crRNA. In E. coli, 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 typically bound to a protein complex (known as Cascade in E. coli[60]).

Interference

This stage requires bound crRNA, as well as cas3 in E. coli[60]. The interference stage targets DNA in most organisms[23][26][51], but RNA targeting has been demonstrated in the case of P. furiosus[33]. Recognition of target DNA is thought to take 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].

In Streptococcus thermophilus, only Cas9 is necessary for CRISPR functionality[74]. However, a specific sequence, known as a proto-adjacent-motif (PAM) was found to be required for interference. The predicted sequence is 5'-NGGNG-3'. This sequence is found several base pairs upstream of the proto-spacer (target DNA). Single base pair mutations in the PAM completely abolish CRISPR interference[74].

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 this is converted into an ~32 bp spacer is unknown.

Cas2 is also involved in integration[30][57], and is a metal dependent endoribonuclease[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 the Cse subtype, regions flanking the proto spacer contain PAMs[37][12][20][28][19], which may be necessary for interference. In general, it is thought that mismatches at positions outside of the spacer sequence allow for targeting, while extended base pairing with the surrounding repeats prevents targeting[37].

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].

Classification of CRISPR systems

For a comprehensive listing of Cas genes, see [1].

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

Type I, II and III systems

This classification takes into account differing mechanisms at all three stages of the pathway[72].

Integration: In type I and II systems, the integration of proto-spacers depends on a proto-adjacent-motif. Cas1 and Cas2 are involved in this stage in all three subtypes.

Expression: The CRISPR locus is transcribed into pre-crRNA. In type I systems, the Cascade complex binds to pre-crRNA, which is then cleaved by the Cas6e or Cas6f. Type II systems use a trans-encoded small RNA (tracrRNA) that binds with the repeat segment of pre-crRNA[59], followed by cleavage by RNase III with Cas9. Cas6 cleaves pre-crRNA in Type III systems. The crRNAs are then transferred to a distinct Cas complex (Csm in subtype III-A and Cmr in subtype III-B).

Interference: In type I systems, the Cascade complex is guided by the crRNA to the target strand. Cas3 then cleaves the DNA. In type II systems, Cas9 directly targets and cleaves the DNA without any additional proteins. Type I and type II systems both probably require a specific PAM for this stage. The Csm or Cmr proteins in type III systems also directly target DNA without additional proteins.

CRISPR resources

  • The [http://crispr.u-psud.fr/crispr/ CRISPR database] analyzes archaeal and bacterial genomes for CRISPR arrays. The sequence of each locus can be displayed. This database is regularly updated.
  • The [http://cmr.jcvi.org/cgi-bin/CMR/shared/GenomePropDefinition.cgi?prop_acc=GenProp0021 J. Craig Venter Institute] CMR genome properties database contains Cas gene information for several hundred genomes. The CMR database is currently without direct funding and is not being actively maintained.
  • [http://crispi.genouest.org CRISPI: a CRISPR Interactive database] analyzes archaeal and bacterial genomes for CRISPR arrays and Cas genes. This database should be used with caution, as many of the purported repeats and spacers are several hundred base pairs, which is in conflict with the literature. Last updated 2008-11-04.