L3 CRISPR/Cas9 Flashcards
CRISPR
Clustered Regularly Interspaced Short Palindromic Repeats
Palindromic
A palindrome is a word, number, phrase, or other sequence of characters which reads the same backward as forward
Cas
CRISPR associated genes
Nucleases
A nuclease (also archaically known as nucleodepolymerase or polynucleotidase) is an enzyme capable of cleaving the phosphodiester bonds between monomers of nucleic acids.
Helicase
Helicases are often used to separate strands of a DNA double helix or a self-annealed RNA molecule using the energy from ATP hydrolysis, a process characterized by the breaking of hydrogen bonds between annealed nucleotide bases
Polymerases
an enzyme which brings about the formation of a particular polymer, especially DNA or RNA.
Polymer
A polymer (/ˈpɒlɪmər/; Greek poly-, “many” + -mer, “part”) is a large molecule, or macromolecule, composed of many repeated subunits.
Locus
the position of a gene or mutation on a chromosome
loci (plural)
Acquisition
(also known as adaptation)
- selection and integration of new spaces from foreign DNA
CRISPR RNA biogenesis
- transcription and enzymatic processing into mature crRNAs
Interference
Cas- mediated cleavage of foreign nucleic acids
crRNA
tracrRNA
crRNA = CRISPR RNA tracrRNA = trans-activating CRISPR RNA
crRNA in the RNA fragment comes from the spacer sequences snipped by the bacteria before. trcrRNA is a gene in the CRISPR system that activate crRNA by maturing it and make together with it what is called gRNA.
Homologous recombination
Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA.
Non-homologous recombination
Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA. NHEJ is referred to as “non-homologous” because the break ends are directly ligated without the need for a homologous template, in contrast to homology directed repair, which requires a homologous sequence to guide repair.
HDR
Homology directed repair
Homology directed repair (HDR) is a mechanism in cells to repair double strand DNA lesions. The most common form of HDR is homologous recombination.
Meganucleases
Meganucleases are endodeoxyribonucleases 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.
Zinc finger nucleases
Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes.
TALEN
Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands).
gRNA
crRNA + tracrRNA
Heterologous expression
Heterologous expression refers to the expression of a gene or part of a gene in a host organism, which does not naturally have this gene or gene fragment.
PAM
Protospacer adjacent motif (PAM) is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease
5’-NGG-3’ for CRISPR/Cas9 type 2
Off-targets
gRNA can target DNA that differs from intended target by up to 5bp
Gene regulation
Gene regulation is the process of controlling which genes in a cell’s DNA are expressed (used to make a functional product such as a protein).
Epigenome editing
histone/chromatin modification
Histone
proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes. They are the chief protein components of chromatin, acting as spools around which DNA winds, and playing a role in gene regulation.
Chromatin
Chromatin is a mass of genetic material composed of DNA and proteins that condense to form chromosomes during eukaryotic cell division. Chromatin is located in the nucleus of our cells.
dCas9
‘dead’ Cas9 or ‘dCas9’ for short, can still tightly bind to dsDNA. This catalytically inactive Cas9 variant has been used for both mechanistic studies into Cas9 DNA interrogative binding and as a general programmable DNA binding RNA-Protein complex.
Nickase Cas9
DNA “nickases” capable of introducing a single-strand cut with the same specificity as a regular CRISPR/Cas9 nuclease
one active site in cas9 mutated
fuse base-converting enzyme to Cas9
- alters single nucleotide in sequence
- When cas9 nicks the other strand, the opposing strand changes
base editing is transient
dCas13
for RNA base editing
dCas13 - RNA binding Cas protien
fuse base-converting enzyme to Cas13
ADARA
Double-stranded RNA-specific adenosine deaminase is an enzyme that in humans is encoded by the ADAR gene (which stands for adenosine deaminase acting on RNA).[5][6]
Adenosine deaminases acting on RNA (ADAR) are enzymes responsible for binding to double stranded RNA (dsRNA) and converting adenosine (A) to inosine (I) by deamination.[7]
