Crispr Flashcards

1
Q

CRISPR acronym

A

Clustered Regulalry Interspaced Palindromic Repeats

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2
Q

what is CRISPR

A

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

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3
Q

SHERLOCK

A

Uses CRISPR-Cas13 to target RNA and is used as a sensitive diagnostic tool

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4
Q

Innate Immunity

A

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

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5
Q

Adaptive immunity

A

A form of immunity that can change or adapt when exposed to new/different pathogens
-Eg. Vaccines

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6
Q

CRISPR background

A

-Discovered when investigating bacterial immunity
-CRISPR is a form of adaptive immunity
-Now been manipulated for genetics and molecular biology research

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7
Q

CRISPR discovery

A

CRISPR loci has been found in 40% of bacteria and 90% of arcahae

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8
Q

CRSIPR spacer sequences

A

identical to fragments of bacteriophage genomes
-Viral sequences within CRISPR loci serve as molecular memory of previous viral attacks

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9
Q

Experimental evidence of CRISPR in adaptive immune system

A

-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.

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10
Q

CRISPR-Cas mechanism principle

A

-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

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11
Q

CRISPR-cas mechanism steps

A

-Spacer acquisition
-crRNA biogenesis
-Target interference

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12
Q

Spacer acquisition

A

-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

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13
Q

crRNA biogenesis

A

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

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14
Q

Target Interference

A

-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

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15
Q

Type I and Type 3 CRISPR-Cas systems

A

Type 1- Cas3 and Type III- Cas10
-Require multi-subunit protein complexes to mediate RNA-guided viral DNA destruction during the interference step

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16
Q

Type II

A

Single Cas9 protein is sufficient
Cas9 plays a role in spacer acquisiton and crRNA biogenesis

17
Q

Cas9 spacer acquisition

A

-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

18
Q

PAM

A

protospacer adjacent motif

19
Q

Cas9 during crRNA biogenesis

A

-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)

20
Q

Modification of Cas systems

A

-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

21
Q

Sequence requirements and components needed for CRISPR

A

-Must have a PAM to be targeted for cleavage
-Needs an sgRNA containing customised crRNA and tracrRNA-derived sequences
-CAs9

22
Q

Method of using CRISPR

A

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

23
Q

DNA damage response pathways

A

-Nonhomologous end-joining (NHEJ)
-Homolog-directed repair (HDR)

24
Q

NHEJ

A

-Ligation of broken fragments
-process is error-prone
-often results in small insertions or deletions (INDELS0 at the repair site

25
Q

HDR

A

-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

26
Q

Cas9 infidelity

A

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

27
Q

CRISPR applications

A

-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

28
Q

CRISPR use in genetic research

A

-Ability to efficiently and quickly delete a gene from the genome
-Reverse genetics: To learn function of gene, delete it and observe consequences

29
Q

Catalytically dead Cas9 (dCas9)

A