CRISPR

Question 1

What are the 3 features of CRISPR?

Answer 1

• 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)

Question 2

CRISPR Typical Structure

Answer 2

• CRISPR Loci
• CRISPR Repeats
• Spacers
• Leader Region
• CRISPR SPacer polarisation and evolution
• Cas genes

Question 3

CRISPR Loci

Answer 3

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

Question 4

CRISPR Repeats

Answer 4

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

Question 5

Spacers

Answer 5

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)

Question 6

Leader Region

Answer 6

o A-T rich sequence
o Site of polarised incorporation: CRSIPR repeat-spacer units are incorporated at this end and contains the CRISPR promoter.

Question 7

CRISPR Polarisation and evolution

Answer 7

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

Question 8

Cas genes

Answer 8

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.

Question 9

CRISPR Classification

Answer 9

• 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)

Question 10

Stages of the CRISPR/CAS System

Answer 10

1. Spacer Acquisition (Immunization/Adaption)
2. cRNA Expression & Processing
3. Interference/Targeting/Immunity

Question 11

Stage 1: Spacer Acquisition

Answer 11

(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.

Question 12

Importance of PAM

Answer 12

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

Question 13

Protospacers & PAM

Answer 13

• 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

Question 14

Stage 2: cRNA Expression & Processing

Answer 14

• 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

Question 15

Stage 3: CRISPR Interference

Answer 15

(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.

Question 16

Cas9 Protein

Answer 16

-	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 section

Question 17

Spacer Acquisition

Answer 17

• 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.

Question 18

cRNA Expression & Processing

Answer 18

• 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.

Question 19

CRISPR Interference/Targeting

Answer 19

• 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.

Question 20

Guide RNA

Answer 20

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.

Question 21

CRISPR RNA Guided Genome Editing

Answer 21

-> Uses NHEJ

1. 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.
2. 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.
3. Cas 9 has double-stranded DNAse activity where two nucleases, RuvC and HNH, will cleave each DNA strand of the target respectively.
4. 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.

Question 22

CRISPR Nuclease RNA guided Genome Editing use HDR for gene insertions

Answer 22

1. Steps 1-3 as for NHEJ (see previous slides) except a repair template is also included upon transformation/transfection.
2. 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.
3. 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

Question 23

CRISPR/Cas Transcription repression

Answer 23

• 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.

Question 24

CRISPR/Cas Transcription activation

Answer 24

• 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

Question 25

Advantages of CRISPR/Cas9 as a Genome-editing tool

Answer 25

• Only requires a Cas9 nuclease and a sgRNA against the target sequence to function as a site-specific nuclease.
• High levels of cutting activity in mammalian cells, particularly at numerous simultaneous targets, (Wang et al., 2013).
• Requirement for an NGG sequence (PAM) makes target design simple
• Fast and cost-effective