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CRISPR-Cas - Coggle Diagram
CRISPR-Cas
Major types
Class 1
Multiprotein effector complexes
Type I, III + IV
Type 1
Signature gene - Cas3
Encodes protein with helicase (unwinding) + endonuclease (cleavage) activities
Other genes from the crRNA effector molecule
Subtypes
1-A - csa3a/csa5 added
1-B - Different organisation
1-C - cas5 replaces cas6
1-D - cas10 fused with cas3 (not type 3)
1-E - Additional cas11
1-F - No cas4
1-G - Different organisation
Type 3
Signature gene - cas10
Encodes multidomain protein
N-terminal cyclase-like domain
Non-enzymatic
Helical domain
Contains Zn-bindin motif
Active cyclase domain
C-terminal \(\alpha\) helical domain
Cmr5 (helical protein)
HD-domain
ssDNase activity
Targets ssDNA/RNA
Large subunit + endonuclease
Cas1 and Cas2
Spacer acquisition from invading nucleic acids (bacteriophage/plasmid)
2 groups based on effector complexes
Csm (Cas subtype Mtube)
A
D
E
F
Target DNA
Cmr (Cas module RAMP (repeat associated mysterious proteins))
B
C
Part of cas10 inactive
Target RNA
Differences in cas11 subunit of effector complex
Type 4
Not properly characterised
DinG helicase necessary for interference
Effector complex
Cas 5
Cas 6
May or may not be part of effector complex
Required for crRNA processing
Cas8
Multiple Cas7 subunits
Class 2
Single-protein effector complexes
All domains needed to make effector molecule in the same gene.
Type II, V + VI
Type 2
Most studied CRISPR related gene
First well-characterised
Signature gene - Cas9
REC - 3 \(\alpha\) helical domains (REC1-3)
NUC
HNH + RuvC nuclease domains
HNH cleaves DNA strand hybridised to crRNA
RuvC cleaves other strand
WED
C-terminal region of wedge
PI
PAM-interacting
BH
Bridge helix
Most suitable for gene editing technologies
Single gene codes for entire effector molecule and endonuclease
tracrRNA
Additional sequence present close to operon
Subtypes
2-A
Cas1,2 and Csn2
S. pyogenes
2-B
Cas1,2,4
F. novidica
Targets endogenous mRNA expression
tracrRNA:scaRNA-cas9 recognises mRNA upon binding of tracrRNA 3' region to target mRNA
Known to target own mRNA
2-C
cas1,2
N. meningitidis
No RNaseIII
Transcription of a short crRNA
Mechanism of interference
Co-processing of Cas9-stabilised tracrRNA:pre-crRNA duplexes
Trimming of crRNA
tracrRNA:crRNA guides Cas9 endonuclease
Site-specific dsDNA breaks in invading DNA
Unknown mechanism
By RNaseIII upon bdinding of tracrRNAanti-repeat to pre-crRNA repeat
Type 5
cpf1 (cas12)
NUC lobe - similar/same to HNH domain
Type 6
Cas13 (C2c2)
RNA-guided RNase providing protection against RNA phage
HEPN1-I and HEPN1-II
Used for diagnostics of pathogens
Cannot cut DNA only RNA
Each type has its own signature gene
Cas1 and cas2 are universal genes but not essential to work.
Cas6 only involved in pre-crRNA processing
Gene order can move around in operon and still make same type
Diff nomenclature in diff publications - dont get confused.
Cmr type- repeat associated mysterious proteins. (RAMP)
Csm based on Mycobacterium
Csm 6 - CRISPR-associated ROssman fold (CARF) domain - regulatory roles.
Genome editing approach
2 major components
gRNA:tracrRNAm+ crRNA
CRISPR-assoc. endonuclease (cas9)
Delivery systems to cells
Microinjected/electroporated
Transfected with plasmids
NHEJ
INDEL
Gene disruption
Wildtyep
Insertion
Deletion
Frameshift
Non-homologous end joining
HDR
+ssDNA
Gene editing
HDR mechanism
Markers introduced
Usually antibiotic resistance gene
Cas9 2 domains
Cas9 nickase
D10A HNH mutant or H840A RuvC mutant
Enhances specificity
2 nicks
Dead Cas9 (dCas9)
DOuble mutant with both D10A HNH and H840A RuvC mutations
Inactive
Activators and repressors cam be attached tod dCas9
CRISPR screening approach
Multiple genes in single assay
GEMMs created by this approach
Personalised therapy
Genomic characterisation of tumours from humans
Gene therapy
Mutation into embryonic stem cells
CRISPR-Cas Systems
Prokaryotic immune systems
Against any invading nucleic acids
e.g. phages and plasmids
Clustered Regularly Interspaced Short Palindromic Repeats
Cas genes are the associated proteins/systems
CRISPR arrays contain short unique spacers separated by repeat sequences of similar size
Each spacer is evolutionarily distinct
Allows for future recognition of that specific foreign genetic material
Spacers are acquired from invading elements
Cas protein complexes recognise PAM sequences
Cas complex cuts short fragment upstream(~2-4nt) to PAM
Incorporates protospacer
CRISPR locus transcribed into pre-crRNA
processed into mature crRNA guides by Cas or non-cas proteins
Interference by degrading
Cas-crRNA complex scans invading DNA for complementary target
Target degraded by Cas nuclease
Cuts 3-4 nt upstream of PAM
Interference
Recognition and acquisition of short sequences from invading DNA
Transcription + processing of crRNAs
By crRNAs-ribonucleoprotein complex or recruitment of Cas nucleases
PAM sequence
Short sequence (~2-6nt)
Sequences are unique
Essential for Cas action
Must start with GGN
Protospacer adjacent motif
Adenine base editors
Using nick CRISPR
Only 1 cut not double stranded
Anything goes wrong, cell restores to original mutation
High product purity
Low indels
SHERLOCK
Not CRISPR-Cas9 but c2c2 (cas13)
DNA - RNA
Colour signal used to detect changes in assay
Dye released when cleavage occurs
Antiviral therapy
Make CD4 and CD8 cells resistant to binding to HIV, stops viral invasion of host cell
CCR5 gene editing
Pathogen evolution and epidemiology
Diphtheria outbreak
PCR used to identify different subtypes
Found epidemic strain of bacterium
Spacer based spoligotyping
Each spacer is a unique evolutionary event
Shown on phylogenetic tree