Transgenics

Question 1

CRISPR

Answer 1

clustered regularly interspaced palindromic repeats

Question 2

CRISPR-Cas was discovered by scientists

Answer 2

trying to understand how bacteria fight viral infection.

Question 3

CRISPR-Cas is a

Answer 3

bacterial immune response system providing resistance to foreign genetic material such as phage DNA.

Question 4

CRISPRs consist of

Answer 4

short base-pair repeats.

Question 5

CRISPR loci contain

Answer 5

interspersed spacers (called protospacers) of viral or plasmid DNA, which come from prior exposures to foreign plasmids or phage.

Question 6

Each protospacer is followed by a

Answer 6

2–6 base-pair segment of spacer DNA, called the protospacer adjacent motif (PAM) 5′-NGG-3′ where N is any nucleotide).

Question 7

Gene editing methods involve the use of specifically engineered DNA-modifying enzymes (nucleases) that allow researchers to create changes in a specific sequence to remove, correct, or replace a defective gene or parts of a gene. Gene editing is based on using different nucleases to create breaks in the genome in a sequence-specific manner. Methods with transcription activator-like effector nucleases (TALENs)/ and zinc finger nucleases (ZFNs) can be used for gene therapy in animal models but have not been successful in humans.

Question 8

CRISPR/Cas genome editing is the

Answer 8

use of engineered (programmable) nucleases to create a double-strand break in DNA in a sequence-specific manner
Can introduce deletions, insertions or substitutions in order to correct or replace a defective gene

Question 9

V1.0 of programmable nucleases:

Answer 9

Meganucleases, Zinc finger nucleases and TALENs

Question 10

V2.0 – CRISPR/Cas [Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / CRISPR-associated protein (Cas)]

Answer 10

Based on an adaptive immune system that evolved in many bacteria and most archaea to protect against infection by phages and plasmids
Modification to make it a genome editing tool led to a Nobel prize for Doudna & Charpentier (2020)
Streptococcus pyogenes - Cas9
Several Cas’es since discovered – also Cas13 that can cut RNA

CRISPR has made most alternative genetic engineering technologies obsolete!!!

Question 11

Question 12

Design of a ____________ is key to CRISPR-Cas technology.

Answer 12

synthetic guide RNA

Question 13

Delivery of both the _______________ is needed to execute the precise DNA excision required

Answer 13

Cas enzyme and the guide RNA

Question 14

Genome editing – CRISPR/Cas9
Components/Requirements

Answer 14

Target gDNA
Cas9 nuclease
CRISPR-RNA (crRNA) & Trans-acting CRISPR-RNA (tracrRNA)
Alt: guideRNA (gRNA)
Protospacer Adjacent Motif (PAM)
DSB repair mechanism - NHEJ or HDR

Question 15

Genome editing – CRISPR/Cas9
Applications

Answer 15

Gene silencing or overexpression
Diagnostics

Question 16

In this example it is important to note the following;
1. The guide RNA is complementary to the your target gene sequence in the genome.
2. The occurrence of the PAM enable the Cas enzyme to execute precise excisions in DNA using the guide RNA as a reference.
3. The technology can be used to create KOs or deliver whole genes (gene replacement).