What are the 3 features of CRISPR?
- Incorporation of Foreign DNA
o CRISPR/Cas system has ability to incorporate short sequences of foreign DNA known as spacers
o Cas proteins incorporate new DNA by cutting it up and adding it to the assay. - Adaptive or Acquired Immunity
o Spacers transcribed into small non-coding RNAs (cRNA which is complementary to incoming phage DNA) which in conjunction with Cas protein complexes target and bind to incoming foreign DNA destroying it.
o Sequence-specific recognition process results in destruction of incoming foreign DNA - Heritable Immunity
o CRISPR/Cas system can readily acquire new spacers (or lose old ones) which allows it to respond dynamically to a viral predator which evolves at higher rates.
o Spacer-derived immunity is inherited by daughter cells (Lamarckian)
CRISPR Typical Structure
- CRISPR Loci
- CRISPR Repeats
- Spacers
- Leader Region
- CRISPR SPacer polarisation and evolution
- Cas genes
CRISPR Loci
o Non-contiguous direct repeats separated by stretches of variable sequences called spacers
o Microbes often contain more than one CRISPR locus
o Loci typically located on chromosome but have been identified on plasmids, phages & prophages
o Have undergone horizontal gene transfer between genomes
o CRISPR systems are divided into different clusters based on their repeat sequences
CRISPR Repeats
o Invariable sequence, vary in length from 23-54 bp
o Most are partially palindromic & can form highly stable secondary structures
o Generally highly conserved within a given CRISPR locus but differs between strains
Spacers
o Contain sequences of ‘captured’ plasmid or phage DNA
o Vary in length from 21-72 bp
o Number of repeat-spacer unit varies in microbes (<50 to 375 units)
Leader Region
o A-T rich sequence
o Site of polarised incorporation: CRSIPR repeat-spacer units are incorporated at this end and contains the CRISPR promoter.
CRISPR Polarisation and evolution
o Linear CRISPR spacer sequences represents a timeline of previous infections and geography (Some phages only occur in specific environments)
o CRISPR loci therefore evolve/adapt in response to viral predation or external plasmid infiltration and are heritable : only known example of Lamarckian evolution
Cas genes
o Cas genes (CRISPR-associated) are often adjacent to CRISPR loci
o Cas genes grouped into three CRISPR/Cas systems: Type I, II, III (and U) and different subtypes
o Encode a large heterogeneous family of proteins: Nucleases, Helicases, Polymerases and Polynucleotide-binding proteins.
CRISPR Classification
- Information processing module:
o New Spacer acquisition
o Cas 1 & 2 proteins - Executive Modules:
o Processing of cRNA and Recognition/Degradation of foreign DNA - Both modules can be happening simultaneously (They are unlinked)
Stages of the CRISPR/CAS System
- Spacer Acquisition (Immunization/Adaption)
- cRNA Expression & Processing
- Interference/Targeting/Immunity
Stage 1: Spacer Acquisition
(Immunization/Adaptation)
- Specific fragments or protospacers (with an adjacent protospacer-associated motif; PAM) of double-stranded DNA from a virus or plasmid are recognised and acquired (integrated) at the leader end of a CRISPR array on host DNA by the action of Cas proteins
- PAM serves as recognition motif required for acquisition. Allows CRISPR system to recognize it.
- Cas 1 & Cas 2 are required (universally present in all CRISPR/Cas systems)
- The Cas 1/Cas 2 complex integrates the protospacer at the leader end of the array
- The CRISPR array consists of unique spacers ; interspaced between repeats; spacers with the most recently acquired DNA are closest to the leader.
Importance of PAM
o PAM is only found on foreign DNA and not on host DNA, therefore allows:
• Spacer selection and acquisition
• Discrimination between self & non-self
• Targeting protospacer for cleavage
o Mismatches at 3’ end of protospacer and/or in PAM allow foreign DNA to escape
Protospacers & PAM
- The foreign DNA corresponding to the spacer DNA is called the protospacer
- This is flanked by a conserved motif (Type I & Type II) called PAM which are 2-5 bp in length
- PAM involved in acquisition (integration), though it is not inserted into the CRISPR array, and also involved in interference/immunity (cleavage of foreign DNA)
- Mutations in PAM allow viruses to escape CRISPR immunity
- Each CRISPR system is very specific in terms of what PAM it will target
Stage 2: cRNA Expression & Processing
- A pre-CRISPR RNA (pre-crRNA) is transcribed as a single transcript from the leader region by RNA polymerase
- The pre-crRNA is further cleaved by Cas proteins into smaller crRNAs (guide RNAs) that contain a single spacer and a partial repeat (hairpin structure)
• 5’ handle has no repeat (just spacer) and 3’ handle has partial repeat
Stage 3: CRISPR Interference
(Targeting/Immunity)
- crRNA containing a spacer that has a strong match to incoming foreign nucleic acid (plasmid or virus) initiates a cleavage event where a multi-protein Cas complex is required.
- There must be near perfect complementarity between crRNA spacer and protospacer target which has an adjacent PAM sequence.
- DNA cleavage interferes with virus replication or plasmid activity and imparts immunity to the host.
- If there is a mismatches between spacer and target DNA or a mutation in the PAM then cleavage is not initiated
NB:
- Easiest way for a phage to escape the CRISPR system, is to have a mutation in the PAM sequence.
- Stages are independent
- For microbial adaptive immunity to be operational all 3 stages must be functional.
- Each stage or process can work independently both mechanistically and temporally
- i.e. A spacer can be acquired from a new phage while interference can occur against a different phage to which previous immunity was acquired.
Cas9 Protein
- Cas9 has double-stranded DNAse activity with two nuclease domains, each of which cleaves one strand of the target DNA
o RuvC-like nuclease at N terminus
o HNH (McrA-like) nuclease domain in the middle sectionSpacer Acquisition
- Cas 1 and 2 proteins are involved in spacer acquisition
- Cas 1 is a homodimeric metal-dependent DNAse that can process ds DNA.
- Cas 1 interacts with other proteins involved in DNA recombination and can repair and resolve Holliday junctions
- It is thought that RecBCD helicase–nuclease complex, which processes DNA double-strand breaks for recombination and degrades foreign DNA, provides these DNA fragments to Cas 1
- Cas 1 recognises these fragments which have a PAM sequence and degrades it further to form protospacers without PAM
- Cas 2 has a RNA recognition domain and has endoribonucleic activity
- Cas 1/Cas 2 integrates protospacers into the leader end of the CRISPR locus, and the repeat sequence is duplicated, maintaining the repeat-spacer-repeat architecture.
cRNA Expression & Processing
- RNA polymerase transcribes pre-crRNA followed by processing into mature crRNAs.
- Processing of the pre-cRNA into cRNA requires trans-encoded small RNA (tra-crRNA) and Cas 9.
- The tra-crRNA shares partial complementarity with CRISPR repeats.
- Cas 9 is required for the tracrRNA to base-pair with the CRISPR repeat region on the pre-crRNA.
- This forms a ds substrate which is cleaved by host RNase III to liberate mature small crRNAs (~24 bp long) comprised of spacer, part of repeat and tracrRNA.
CRISPR Interference/Targeting
- Protospacers in type II systems are flanked by a 3′ PAM
- The cRNA serves as a ‘guide’ (guide RNA) to allow specific base-pairing between exposed cRNA within Cas 9 and the protospacer on the foreign DNA
- crRNA:tracrRNA drives Cas 9 conformational changes that directs target DNA binding in a PAM-dependent manner.
• If tracrRNA is removed, there will be no targeting of foreign DNA. Same thing if PAM is removed. - Mature crRNA, together with Cas 9, interferes with matching invasive ds-DNA by homology-driven nuclease cleavage within the protospacer sequence.
- Cleavage done by RuvC and HNH nucleases within Cas 9
- Cas 9 nucleases will cut 3-4 nucleotides upstream of the PAM sequence.
Guide RNA
sgRNA
- Linkage of tracrRNA to crRNA opens up gene editing to all cells.
- Implications:
• Human genome is sequenced, so you can look for a specific gene you want to target and look for a PAM sequence next to it
• For Cas9 PAM motif = N(any base)GG
• Design spacer complementary to DNA sequence of choice
• Attach Cas9 to sgRNA
• RNA attaches to target DNA sequence, Cas9 dsDNA cuts
• DNA can be deleted causing frameshift, or new DNA can be inserted.
CRISPR RNA Guided Genome Editing
-> Uses NHEJ
- A single guide RNA (sgRNA) consists of a crRNA for the target region attached to the tracrRNA.
These sgRNA’s must be designed for targeting taking the PAM sequence (5’NGG3’) on the target DNA into consideration and synthesised.
sgRNAs & Cas 9 can be introduced into different types of cells using appropriate delivery systems. - The sgRNA together with Cas 9 will bind to the target region (protospacer) of the genome if there is a PAM sequence (5’NGG3’) at the 3’ end of the target region.
- Cas 9 has double-stranded DNAse activity where two nucleases, RuvC and HNH, will cleave each DNA strand of the target respectively.
- Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA. This pathway is referred to as “non- homologous” because the break ends are directly ligated without the need for a homologous template. This creates a deletion in the target region or gene knockout (InDel) as it leads to frameshifts and/or premature stop codons, effectively disrupting the open reading frame (ORF) of the targeted gene.
CRISPR Nuclease RNA guided Genome Editing use HDR for gene insertions
- Steps 1-3 as for NHEJ (see previous slides) except a repair template is also included upon transformation/transfection.
- The Homology Directed Repair (HDR) pathway can be used to insert new genes or change the function of an existing gene.
a. Requires the presence of a repair template, which is used to fix the double strand break (DSB).
b. If you put another gene onto that repair template, then you could insert a new gene. - HDR faithfully copies the sequence of the repair template to the cut target sequence. Specific nucleotide changes can be introduced into a targeted gene by the use of HDR with a repair template
CRISPR/Cas Transcription repression
- Nuclease-deficient Cas9 (dCas9) in complex with specific sgRNAs bind target DNA to inhibit transcriptional initiation, elongation and the binding of transcription factors.
• Because it doesn’t have nuclease activity, it can target and bind to a region but can’t cut it.
• It blocks RNA Pol II from binding.
CRISPR/Cas Transcription activation
- dCas9 are fused to domains that assist activation, such as the omega subunit of RNA Pol or multiples of VP16 in eukaryotes,
- This can promote the upregulation of target genes
• RNA Pol will see activator and bind to it
