Question

can someone describe IN DETAIL the steps involved in the defence mechanism in bacteria with crispr

(adaptation, biogensis, interference)

please provide all details, make it long and very specific

Answer 1

• CRISPR is an adaptive defence system i.e. Clustered Regularly Interspaced Short Palindromic Repeats
• steps of CRISPR-Cas defence mechanism

1. ADAPTATION

• The first step is adaptation and acquisition. in this phase, genetic material of invading phage is incorporated into CRISPR-Cas system.
• Key proteins- Cas1 ans Cas2- , have nuclease activity ,plays crucial role, ubiquitous in nature , doesnot potentiate spacer acquisition on their own but mutation in these proteins leads to spacer acquisition impossible and overexpression leads to improvement in spacer acquisition.
• Cas1 and Cas 2 forms heterohexameric protein complex comprising of 1 Cas2 dimer and 2 Cas1 dimers( Cas1a, 1a', Cas1b, 1b'). complex performs dual function i.e.the excision of protospacer DNA and its incorporation into CRISPR segment. PAMs- Protospacer Adjacent motifs determine spacer alignment in type 1 and type 2 systems.
• Type 1 system is for ssDNA in which Cas1 binds to complementary sequence of PAM.
• Type 2 system is for dsDNA in which Cas 9 bind to PAM.
• Cas1a and Cas1a' recognises PAM and Tyr residue in Cas1 limits the central dsDNA to length of 23 nucleotide. then Cas2 fron comlex overhangs 7 nucleotides at each 3' end from which 3 nucleotides are cleaved by nCas1 domains forming to 3'-OH group and forms mature protospacer of 33 nucleotides. then Cas1- Cas2 complex act as integrase and leads to integration protospacer DNA into CRISPR by nucleophilic attack at both 5'end and forms an extended CRISPR array with a new spacer . that is how adaptation occurs. some systems also use Cas 4 nuclease but widely used are Cas1 and Cas2.

2. BIOGENESIS is definesd as trascription and processing of cas genes and CRISPR into small crRNAs

STEPS-

• In this, CRISPR locus is cleaved by Cas proteins or cellular ribonucleases which results in the formation of mature crRNAs( it contains repeat regions flanked on single spacer sequence). different system involves different proteins to convert pre-crRNA into mature cr-RNA.
• TYPE -1 system- Cas 6 is involved and spacer forms stem-loop structure and converts pre-crRNA into mature cr-RNA.
• TYPE-2 system - Cas9, RNase III , small trans- activating RNAs are involved. in this dsRNA repeats with alternative ss spacers are formed in which Cas9 stabilises small- trans activating RNAs, and then cleavage of pre-crRNA by RNase-III and furthur processed at 5' end to form  mature cr-RNA through a ruler based mechanism.
• TYPE-3 system is same as TYPE-2 system , the only difference is of last trimming which oocurs at 3'end to form mature cr-RNA.
• TYPE-4 system is rare. in this, Csf1, Cas5 and Cas7 are involved to convert pre-crRNA into mature cr-RNA.
• TYPE-5 - Cas12 is involved to convert pre-crRNA into mature cr-RNA.
• TYPE-6- Cas 13 is involved to convert pre-crRNA into mature cr-RNA.

3. INTERFERENCE

• It is used to enable the silencing of foreign genetic material with the help of mature cr-RNA.
• TYPE-1- cascade system of different Cas proteins in involved formed of multiprotein backbone with Cas proteins linked to crRNA which recognises the PAM site and unwinds DNA forming the pair of crRNA with homologous DNA strand which leads to the formation of R- loop formation and the Cas3 comes cleaves the free ssDNA. in this full degradation might not happen . for complete degaradation, Cascade- independent Cas3 nuclease activity is required.
• TYPE-2- both Cas9 and small- trans activating RNAs are involved. Cas 9 acts as endonuclease. crRNA and small- trans activating RNAs are involved and forms duplex due to complementarity to spacer sequences of crRNA. this duplex leads to activation of Cas9 by conformational change which finds foreign genetic elements for PAM site . upon identification, dsDNA is unwinded and crRNA bind to ssDNA and R- loop is formed which is furthur processed by Cas9, RuvC nad HNH at nucleotide upstream of PAM.
• TYPE-3- same as TYPE-1, involves cascade system. Cas10 , Cas7 and Cms in subtype III-A and Cmr in subtype III-b are involved. Cas10 acts on both DNA and RNA. cascade complex binds to ssRNA which leads to Cas10 mediated cleavage of dsDNA and Cas7 mediated cleavage of ssRNA. and then Cas10 activates Csm6 and degrade foreign transcripts.
• TYPE-V- Cas 12 is involved and the process is same as Cas9 with the difference that it does not depends upon small trans- activating RNAs. they primarily depend upon crRNA.
• TYPE-VI- Cas13 is involved. species dependent PFS( protospacer flanking site) analogous to PAM in target DNA activate Cas 13 and binding to crRNA leads to conformational change in Cas13 and leads to ssRNA pairing.then Cas13 RNase activity is increased by catalytic sites of both HEPN domains.