Lecture 7: CRISPR and meganucleases Flashcards
(33 cards)
_______: Characterized by a large recognition site (double-stranded
DNA sequences of 12 to 40 base pairs)
* as a result this site generally occurs only once in any given
genome
meganucleases (endonucleases)
_______ are therefore considered to be the most
specific naturally occurring restriction enzymes
Meganucleases
_______ are found in a large number of organisms – fungi, yeast, algae
and some plants
meganucleases
where can meganucleases be expressed?
in different compartments of the cell –
the nucleus, mitochondria or chloroplasts
In nature, _______ are encoded by mobile genetic
elements (e.g. transposons), introns or inteins (a segment of a
protein that is able to excise itself and join the remaining
portions with a peptide bond in a process termed protein
splicing)
meganucleases
self-splicing protein!!
Several families of homing _______ was of interest as a
valuable tooi for the study of genomes and genome engineering
endonucleases
there are a bunch of different kinds/uses of endonucleases… generally all are….. though
restriction endonucleases
why would we want to use endonucleases for genome editing?
Mutational analysis (genotype-phenotype)
Can influence translation (eg, RNA interference – RNA via
siRNA)
why is it better to mutate the gene itself- or its promoter? when we would historically randomly mutagenize an organism
impractical for complex organisms- super tedious to just search for what changed!!
IN GENERAL
* Editing or regulation of genomic information at the DNA level
requires the action of a molecular machine composed of two
parts: what are they?
- DNA-binding domain that mediates sequence-specific DNA
recognition and binding (20-30 bp motif) - An effector domain that enable DNA cleavage near the binding
site (cutting)
T/F: Creating a double stranded break (DSB) by using sequence-
specific endonucleases can stimulate the DNA repair
pathway and increase the rate of gene modification at
desired sequence
true!!
what does Crispr-Cas mean?
Clustered Regularly Interspaced Short Palindromic Repeats
(DNA) and
CRISPR associated proteins (CAS)
T/F: In nature CRISPR-Cas serve as an adaptive immune mechanism
true
In many bacteria and most archaea, clustered, regularly
interspaced short palindromic repeats (CRISPRs) are involved in
an interference pathway that protects cells from ____ and _____
bacteriophages
and conjugative plasmids
CRISPR sequences provide an adaptive, heritable record of past
infections and express ______ — small RNAs that target
invasive nucleic acids
CRISPR RNAs
______ is one of several sequence-directed genetic interference pathways
that control gene expression and preserve genome integrity
CRISPR-Cas
what are the features of a CRISPR loci?
Typically, clustered, regularly interspaced short palindromic repeats (CRISPRs,
white boxes) are preceded by a leader sequence (black box) that is AT-rich but
otherwise not conserved. The number of repeats can vary substantially, from a
minimum of two to a few hundred.
A set of CRISPR-associated (cas) genes immediately precedes or follows the
repeats. These genes are conserved, can be classified into different families and
subtypes, and encode the protein machinery responsible for CRISPR activity.
the repeat length of a CRISPR loci is restricted to…
23 to 50 nucleotides
Repeats are
separated by similarly sized, non-repetitive spacers that share
sequence identity with fragments of plasmids and bacteriophage genomes and specify the targets of CRISPR interference
what are the three phases of CRISPR function?
Acquisition of new spacers into CRISPR arrays (from recent defeat of a phage, etc.)
Expression and processing of CRISPR RNAs (crRNAs)
CRISPR interference
In the _____ phase, CRISPR loci incorporate additional spacers to
programme their activity against invading plasmids and phages.
first
incorporating additional spacers into the CRISPR loci allows the cell to…
adapt rapidly to the invaders present in the
environment and is referred to it as the ‘adaptation’ phase of CRISPR
function
The information stored in spacers is then used to repel invaders
during the ‘_______’ phase of CRISPR interference
defence
mechanism of spacer acquisition likely uses the nuclease activity of
_________ to generate short fragments of invading
DNA, and is selected by containing short 2-6 bp protospacer adjacent
motif (PAM) sequences (PAM is not integrated)
CRISPR-associated 1 (Cas1)
The addition of new spacers is thought to involve an integration or
conversion event and occurs at the _______ end of the cluster
leader/proximal
