Lecture Notes on the CRISPR/Cas module (MCB2023S)

CRISPR
CRISPR.......................................................................................................................................................... 1
OVERVIEW.................................................................................................................................................... 2
CONCEPT BEHIND CRISPR/CAS:..............................................................................................................................2
TYPICAL STRUCTURE...............................................................................................................................................3
STAGES OF THE CRISPR/CAS SYSTEM............................................................................................................. 4
STAGE 1: SPACER ACQUISITION (IMMUNIZATION/ADAPTATION).....................................................................................4
Protospacers & PAM:......................................................................................................................................4
STAGE 2: CRNA EXPRESSION & PROCESSING..............................................................................................................5
STAGE 3: CRISPR INTERFERENCE (TARGETING/IMMUNITY)...........................................................................................5
MECHANISMS OF ACTION............................................................................................................................. 7
TYPE II SYSTEM CRISPR/CAS 9...............................................................................................................................7
Spacer Acquisition..........................................................................................................................................7
cRNA expression & processing........................................................................................................................7
CRISPR Interference/Targeting.......................................................................................................................7
GENOME EDITING......................................................................................................................................... 9
Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza...............................9
Guide RNA – sgRNA........................................................................................................................................9
CRISPR/CAS NUCLEASE RNA-GUIDED GENOME EDITING USING NHEJ.........................................................................10
CRISPR/CAS NUCLEASE RNA-GUIDED GENOME EDITING USING HDR FOR GENE INSERTIONS...........................................10
CRISPR/CAS GENE REGULATION...........................................................................................................................11
Transcription repression...............................................................................................................................11
Transcription activation................................................................................................................................11
ADVANTAGES OF CRISPR/CAS 9 AS A GENOME-EDITING TOOL....................................................................................11

, CRISPR
Overview
Concept behind CRISPR/CAS:
- Part of the adaptive microbial immune system
- Clustered Regularly Interspaced Short Palindromic Repeats
- Family of DNA repeats found in:
o 90% of Archaea
o 40% of Bacteria
- Initially thought of as junk DNA
- Is a ‘memory of past genetic aggressions’
o i.e. showed what viruses and plasmids and phages the bacteria had been
exposed to in the past.
- Provides adaptive immunity – the removal/addition of CRISPR spacer sequences can
affect resistance to phage attack.

- CRISPR array associated with cas genes
- Array:
o Leader:
 AT rich
 Contains CRISPR promoter
o Spacers:
 Variable sequences of similar length
 17-100bp; 36bp average
o Repeats:
 Invariable sequence except the terminal repeat
 Partially palindromic
 23-50bp
 Forms stem loop

Features:
- 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.