Crispr Flashcards
CRISPR acronym
Clustered Regulalry Interspaced Palindromic Repeats
what is CRISPR
It is the hallmakr of the bacterial defence system and is part of their adaptive immune system
-It is used by researcehrs in modifying genes permanently in living cells and organsisms
-Allows for the correction of mutations at precise locations to treat genetic causes of diseases
SHERLOCK
Uses CRISPR-Cas13 to target RNA and is used as a sensitive diagnostic tool
Innate Immunity
An interna system that can be used to defend against foregin pathogens
-restricitons enzymes are involved in this
-RE coding sequences are fixed andd protein structure remains stable for long periods of time
Adaptive immunity
A form of immunity that can change or adapt when exposed to new/different pathogens
-Eg. Vaccines
CRISPR background
-Discovered when investigating bacterial immunity
-CRISPR is a form of adaptive immunity
-Now been manipulated for genetics and molecular biology research
CRISPR discovery
CRISPR loci has been found in 40% of bacteria and 90% of arcahae
CRSIPR spacer sequences
identical to fragments of bacteriophage genomes
-Viral sequences within CRISPR loci serve as molecular memory of previous viral attacks
Experimental evidence of CRISPR in adaptive immune system
-Exposed S.thermophillus to specific phage
-Bacterial cells that survived became resistant to that phage but not others
-Resistant bacteria possessed new spacers within their CRISPR loci with an exact sequence match to portions of the phage genome they were exposed to
-Deletion of new spacer abolished phage resistance
-Experimental insertions of new viral sequence-derived spacers into the CRISPR loci of sensitive bacteria rendered them resistant.
CRISPR-Cas mechanism principle
-RNA guided destruction of invading DNA (nucleotuides)
-Adaptive immunity is also dependant on a set of adjacent CRISPR-associated (cas) genes
-Cas genes encode Cas proteins that function as DNases and RNases
CRISPR-cas mechanism steps
-Spacer acquisition
-crRNA biogenesis
-Target interference
Spacer acquisition
-Invading phage DNA is cleaved into smaller fragments known as protospacers, which are then inserted into CRISPR loci to become new spacers
-When new spacers are added to the CRISPR locus, repeat sequences are duplicated such that each spacer is flanked by repeats in each side
crRNA biogenesis
CRISPR loci are transcribed starting att the promoter within the leader, into long RNA transcripts known as pre-crRNA
-Processed into short CRISPR-derived RNAs (crRNAs), each containing a single spacer flanked on both sides by repeat sequences
Target Interference
-Mature crRNAs associated with Cas nucleases, or nuclease complexes, and recruit them to complementary sequences in invading phage DNA
-Cas nucleases then cleave the viral DNA, thus neutralising infection
Type I and Type 3 CRISPR-Cas systems
Type 1- Cas3 and Type III- Cas10
-Require multi-subunit protein complexes to mediate RNA-guided viral DNA destruction during the interference step
Type II
Single Cas9 protein is sufficient
Cas9 plays a role in spacer acquisiton and crRNA biogenesis
Cas9 spacer acquisition
-Cleaves invading viral DNA
-Cas9 selects prtospacer sequences flanked by a PAM defined by the sequence 5’-NGG-3’
-After protospacer cleavage, the Cas1/Cas2 complex integrates the DNA into a CRISPR locus
PAM
protospacer adjacent motif
Cas9 during crRNA biogenesis
-Noncoding RNA called transactivating crRNA (tracrRNA) binds to crRNA repeat
-Complex is then bound by Cas9 and cleaved into mature crRNA/tracrRNA duplexes by RNase that specifically recognises double-stranded RNA (RNase III)
Modification of Cas systems
-crRNA and tracrRNA can be modified artificially to produce a hybrid single guide RNA (sgRNA)
-A 20-nucleotide-long targeting sequence of a crRNA joined to minimal sequences of the tracRNA necessary for Cas9 function
-The sgRNA and Cas9 gene sequene may be incorporated into an expression vector so that they may be expressed and utilised in eukaryotic cells for genome editing
Sequence requirements and components needed for CRISPR
-Must have a PAM to be targeted for cleavage
-Needs an sgRNA containing customised crRNA and tracrRNA-derived sequences
-CAs9
Method of using CRISPR
Engineered plasmid expression vectors carrying encoding Cas9 and an sgRNA witha specific DNA targeting sequence introduced into mammalian cell culture
-CRISPR-Cas9 cut precise sequence and forms a double stranded break
-Endogenous DNA repair mechanims fixed break but created indels or added new donor sequence
DNA damage response pathways
-Nonhomologous end-joining (NHEJ)
-Homolog-directed repair (HDR)
NHEJ
-Ligation of broken fragments
-process is error-prone
-often results in small insertions or deletions (INDELS0 at the repair site
HDR
-Uses an undamaged homologous chromosome or sister chromatid as a template to correctly repair broken chromosome
-Can be tricked into using an artificial donor template to make complex substitutions, deletions, or additions
-Less error prone
Cas9 infidelity
It can also cut off-target sites in genome
-Off-target edits may be due to an sgRNA having more than one perfect match in genome
-Improving the specificity of CRISPR edits is important for safety of medical-applications of this technology
CRISPR applications
-Basic genetic research
-Used in biotech for cost-effective production of genetically modified crops that have enhanced nutritional value, etc
-Clinical uses for treatment of genetic disorders that involve single gene mutations
-Must be used carefully due to off-target effects
CRISPR use in genetic research
-Ability to efficiently and quickly delete a gene from the genome
-Reverse genetics: To learn function of gene, delete it and observe consequences
Catalytically dead Cas9 (dCas9)
