L3 CRISPR/Cas9

Question 1

CRISPR

Answer 1

Clustered Regularly Interspaced Short Palindromic Repeats

Question 2

Palindromic

Answer 2

A palindrome is a word, number, phrase, or other sequence of characters which reads the same backward as forward

Question 3

Cas

Answer 3

CRISPR associated genes

Question 4

Nucleases

Answer 4

A nuclease (also archaically known as nucleodepolymerase or polynucleotidase) is an enzyme capable of cleaving the phosphodiester bonds between monomers of nucleic acids.

Question 5

Helicase

Answer 5

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

Question 6

Polymerases

Answer 6

an enzyme which brings about the formation of a particular polymer, especially DNA or RNA.

Question 7

Polymer

Answer 7

A polymer (/ˈpɒlɪmər/; Greek poly-, “many” + -mer, “part”) is a large molecule, or macromolecule, composed of many repeated subunits.

Question 8

Locus

Answer 8

the position of a gene or mutation on a chromosome

loci (plural)

Question 9

Acquisition

Answer 9

(also known as adaptation)

• selection and integration of new spaces from foreign DNA

Question 10

CRISPR RNA biogenesis

Answer 10

• transcription and enzymatic processing into mature crRNAs

Question 11

Interference

Answer 11

Cas- mediated cleavage of foreign nucleic acids

Question 12

crRNA

tracrRNA

Answer 12

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.

Question 13

Homologous recombination

Answer 13

Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA.

Question 14

Non-homologous recombination

Answer 14

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.

Question 15

HDR

Answer 15

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.

Question 16

Meganucleases

Answer 16

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.

Question 17

Zinc finger nucleases

Answer 17

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.

Question 18

TALEN

Answer 18

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).

Question 19

gRNA

Answer 19

crRNA + tracrRNA

Question 20

Heterologous expression

Answer 20

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.

Question 21

PAM

Answer 21

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

Question 22

Off-targets

Answer 22

gRNA can target DNA that differs from intended target by up to 5bp

Question 23

Gene regulation

Answer 23

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).

Question 24

Epigenome editing

Answer 24

histone/chromatin modification

Question 25

Histone

Answer 25

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.

Question 26

Chromatin

Answer 26

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.

Question 27

dCas9

Answer 27

‘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.

Question 28

Nickase Cas9

Answer 28

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

Question 29

dCas13

Answer 29

for RNA base editing

dCas13 - RNA binding Cas protien

fuse base-converting enzyme to Cas13

Question 30

ADARA

Answer 30

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]