• Home
• Team
  • Official Team Profile
  • Photos
• Project
• Lab
  • Protocols
• Parts
• Modeling
• Notebook
  • March
  • April
  • May
  • June
  • July
• Safety
• Attributions

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.

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]:

• These 5 genes combine to form the cascade complex^[60]
• Their transcription is repressed by H-NS^[67], and de-repressed by leuO^[45] or baeR^[54]
• In particular, casE cleaves pre-crRNA^[23], and casA and casB can be omitted without affecting crRNA generation^[60].

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: