Crispr/Cas
Lecture 1
Microbial Defense Mechanisms Against ‘Foreign’ DNA
• Need to maintain gene$c integrity against mobile gene4c elements (MGEs = phages,
plasmids, transposons)
o Exogenous DNA can enter via
§ Conjuga4on
§ Transduc4on
§ Transforma4on
• Ability to discriminate between foreign and self DNA
• Against viruses:
o Prevent adsorp4on
o Blocking of viral DNA injec4on
o Abor4ve infec4on
• Against target DNA:
o Restric4on
o Sugar nonspecific nucleases
• Against MGEs:
o Adap4ve microbial immune system
Adap:ve Microbial Immune System
• Clustered regularly interspaced short palindromic repeats
o 90% of archaea
o 40% of bacteria
§ Problem is self immunity!
• Sequences similar to own DNA can be incorporated and lead to
aUack of own DNA
• Homology between CRISPR repeats and extrachromosomal elements (viruses and
plasmids)
• Adap4ve! à there is a memory of past gene4c aggressions
o Found that bacteria could be “vaccinated” against phage aUack through
making edits to the CRISPR sequences
Typical Structure of CRISPR/Cas Systems
• Cas genes
o Can surround or be before the CRISPR array region
o O\en adjacent to CRISPR
o Grouped into 3 systems (Type 1,2,3 and U) + different substypes
o Encode large heterogenous family of proteins
§ Nucleases
§ Helicases
§ Polymerases
, § Polynucleo4de binding proteins
• CRISPR array
o Leader
§ Site of polarized incorpora:on of exogenous DNA
• Every new sequence is added to the same point
• Linear CRISPR spacer sequences represent a 4meline of
previous infec4ons + geography
§ Has a promotor region where RNA pol binds for transcrip4on
§ Site of spacer inser4on
§ AT rich sequence
o Spacer
§ Contain captured/foreign DNA
§ Spacers are separated by repeats
• Repeats have cleavage points in the stem loop
• Average 36 bp long (17-100 bp)
o Repeat
§ Hairpin loop
§ Par4ally palindromic sequence
§ 23-50 bp
§ Highly stable secondary structures
§ Highly conserved within a given CRISPR sequence but differs between
strains
o Terminal repeat
*bacteria have different crispr/cas systems in them (repeats are unique in their systems)
CRISPR Loci à
• Non-con4guous direct repeats separated by stretches of variable sequences called
spacers
• Microbes o\en have >1 locus
• Typically located on chromosome but seen on plasmids, phages and prophages
o Have undergone HGT between genomes
• Divided into different clusters based on repeat sequences
• Adapt + evolve in response to viral preda4on or external plasmid infiltra4on
o heritable
Features of CRISPR/CAS system
• Incorpora4on of foreign DNA (spacers)
• Adap4ve/acquired immunity
- Spacers transcribed into small non-coding RNAs
- Cas protein complexes in conjunc4on with RNAs target + bind to foreign DNA
- Sequence specific recogni4on process results in destruc4on of incoming foreign
DNA
o Lamarckian evolu4on
• Heritable immunity
- CRISPR/cas can readily acquire new spacers or lose old ones (dynamic)
- Allows for response to viral predators evolving at higher rates
Lecture 1
Microbial Defense Mechanisms Against ‘Foreign’ DNA
• Need to maintain gene$c integrity against mobile gene4c elements (MGEs = phages,
plasmids, transposons)
o Exogenous DNA can enter via
§ Conjuga4on
§ Transduc4on
§ Transforma4on
• Ability to discriminate between foreign and self DNA
• Against viruses:
o Prevent adsorp4on
o Blocking of viral DNA injec4on
o Abor4ve infec4on
• Against target DNA:
o Restric4on
o Sugar nonspecific nucleases
• Against MGEs:
o Adap4ve microbial immune system
Adap:ve Microbial Immune System
• Clustered regularly interspaced short palindromic repeats
o 90% of archaea
o 40% of bacteria
§ Problem is self immunity!
• Sequences similar to own DNA can be incorporated and lead to
aUack of own DNA
• Homology between CRISPR repeats and extrachromosomal elements (viruses and
plasmids)
• Adap4ve! à there is a memory of past gene4c aggressions
o Found that bacteria could be “vaccinated” against phage aUack through
making edits to the CRISPR sequences
Typical Structure of CRISPR/Cas Systems
• Cas genes
o Can surround or be before the CRISPR array region
o O\en adjacent to CRISPR
o Grouped into 3 systems (Type 1,2,3 and U) + different substypes
o Encode large heterogenous family of proteins
§ Nucleases
§ Helicases
§ Polymerases
, § Polynucleo4de binding proteins
• CRISPR array
o Leader
§ Site of polarized incorpora:on of exogenous DNA
• Every new sequence is added to the same point
• Linear CRISPR spacer sequences represent a 4meline of
previous infec4ons + geography
§ Has a promotor region where RNA pol binds for transcrip4on
§ Site of spacer inser4on
§ AT rich sequence
o Spacer
§ Contain captured/foreign DNA
§ Spacers are separated by repeats
• Repeats have cleavage points in the stem loop
• Average 36 bp long (17-100 bp)
o Repeat
§ Hairpin loop
§ Par4ally palindromic sequence
§ 23-50 bp
§ Highly stable secondary structures
§ Highly conserved within a given CRISPR sequence but differs between
strains
o Terminal repeat
*bacteria have different crispr/cas systems in them (repeats are unique in their systems)
CRISPR Loci à
• Non-con4guous direct repeats separated by stretches of variable sequences called
spacers
• Microbes o\en have >1 locus
• Typically located on chromosome but seen on plasmids, phages and prophages
o Have undergone HGT between genomes
• Divided into different clusters based on repeat sequences
• Adapt + evolve in response to viral preda4on or external plasmid infiltra4on
o heritable
Features of CRISPR/CAS system
• Incorpora4on of foreign DNA (spacers)
• Adap4ve/acquired immunity
- Spacers transcribed into small non-coding RNAs
- Cas protein complexes in conjunc4on with RNAs target + bind to foreign DNA
- Sequence specific recogni4on process results in destruc4on of incoming foreign
DNA
o Lamarckian evolu4on
• Heritable immunity
- CRISPR/cas can readily acquire new spacers or lose old ones (dynamic)
- Allows for response to viral predators evolving at higher rates
