test 2 Flashcards
What does CRISPR mean
Clustered Regularly Interspaced Palindromic Repeats
What is Cas9
CRISPR Associated Protein 9-A bacterial endonuclease that cuts the DNA (making a double stranded break) at a specific site within a target sequence.
What is Single Guide RNA (sgRNA):
An engineered form of guide RNA (~100 nt long) made of the fusion of 2 RNAs (that would occur separately in nature) which forms a complex with the Cas9 endonuclease
What is the Guiding Region of sgRNA
The guiding region is adjusted by scientists to cut different targets.
What is the Scaffold Region of the sgRNA
Transactivating CRISPR RNA (tracRNA) in nature which forms a multi-hairpin loop structure (or scaffold) that tightly binds a Cas9 crevice
The scaffold region sequence is the same for nearly all sgRNAs
What is Protospacer Adjacent Motif (PAM):
A sequence motif immediately downstream of the protospacer protein in the Cas9 recognition sequence that is required for proper Cas9 function
How does PAM recognize Cas9
a) Cas9 recognizes the PAM sequence (which is 5’-NGG where N is any of the 4 DNA nucleotides, and G is guanine)
b) When Cas9 binds to the PAM, it separates the DNA strands of the adjacent sequence to permit the sgRNA to bind.
c) If the sgRNA is complementary to the sequence, Cas9 will cleave the DNA
What is CRISPR-Cas9 gene editing?
CRISPR-Cas9 is a prokaryotic immune system to protect bacteria from viral invaders.
Steps of CRISPR-Cas9 gene editing
- Cas9 binds to an sgRNA
- The Cas9-sgRNA complex binds to a PAM site on the target DNA
- The guiding region of the sgRNA binds to the target DNA sequence
- Cas9 makes a double stranded break in the DNA 3 base pairs upstream of the 5’-NGG PAM sequence
- The complex releases from the DNA
What is the LacZ gene
- LacZ is part of the Lac operon
- Specifically, LacZ encodes the enzyme β-galactosidase
Non-homologous End Joining (NHEJ)
(1) The ends of the double strand breaks are annealed back together by enzymes
(2) Error prone-can result in the insertion or deletion of bases, causing an interruption to normal gene function
Homologous Repair (HR)
(1) Enzymes repair the damage by using a homologous sequence as a template
(2) HR is less error prone than NHEJ
In our experiment, when E. coli was grown in the presence of X-gal, hydrolysis of X-gal would result in the production of blue pigment.
Wild type
Polymorphic
The presence of two or more different forms of an allele or trait-one version is not necessarily deleterious to the other(s)
VNTR
Variable Number Tandem Repeat-Longer repetitive sequences present throughout the genome
