Learning Outcomes - Week 12 - DNA Editing Using CRISPR Flashcards
Understand that various DNA editing technologies exist. Give examples and explain each briefly
E.g., (restriction) endonucleases,
zinc finger nucleases, CRISPR
Endonucleases
* Restriction enzymes are great for cutting DNA in the lab
* Cloning of genes into plasmids, etc.
* Not good if we want to make precise edits to genomic DNA in the cell
* Recognition sequences are short – typically 4 to 8 bp.
* Cutting sites present all over human genome of 3 billion bp
* Precise gene editing requires programmable nucleases
* Longer recognition sequences for more specificity across genome
(See image for what CRISPR is)
CRISPR/Cas9 is a powerful…?
gene editing technology
What is the mechanism of CRISPR/Cas9 technology?
The mechanism of CRISPR/Cas-9 genome editing can be generally divided into three steps: recognition, cleavage, and repair. The designed sgRNA directs Cas-9 and recognizes the target sequence in the gene of interest through its 5ʹcrRNA complementary base pair component.
- What is CRISPR derived from?
- Why is CRISPR so good?
- Which components that are critical for successful genome editing using CRISPR/Cas9
- The CRISPR-Cas9 system derived from Streptococcus
pyogenes (Sp) has become the gene editing system of
choice - Faster, cheaper, more accurate than previous gene editing systems
- CRISPR RNAs contain approx. 20 base recognition sequence
* Spacer sequence - binds to protospacer in DNA
* Programmable – change the spacer sequence to change specificity
of target
* PAM sequence – Protospacer Adjacent Motif
* For Cas9 = NGG (N = A, C, G or T)
* Other Cas nucleases use different PAM sequences
* E.g. Cas12a = TTTV (V= A, C or G)
Be able to mention a few general CRISPR experimental designs/ways in which
CRISPR can be used to edit the genome.
Explain each
mutation, deletion (including
gene deletion/knock-out), insertion
CRISPR/Cas9 works by cutting a DNA sequence at a specific genetic location and deleting or inserting DNA sequences, which can change a single base pair of DNA, large pieces of chromosomes, or regulation of gene expression levels
Know that CRISPR is frequently used for…? Example?
- genome-wide screening :
- Genome-wide CRISPR-Cas9 knockout screens aim to elucidate the relationship between genotype and phenotype by ablating gene expression on a genome-wide scale and studying the resulting phenotypic alterations. - genome imaging:
- CRISPR-Cas9 based imaging techniques allow scientists to track genomic loci of interest in real time. Cas9 is modified to contain an imaging probe (i.e., fluorescent protein) and introduced into cells along with a guide RNA. (See image for slide) - chromatin dynamics:
- Chromatin Regulation and Dynamics integrates knowledge on the dynamic regulation of primary chromatin fiber with the 3D nuclear architecture, then connects related processes to circadian regulation of cellular metabolic states, representing a paradigm of adaptation to environmental changes.
Able to mention a few general CRISPR applications in research (e.g., …?) Explain
gene knock-out, gene knock-in, epigenome regulation (shown in images)
Able to think broadly about how CRISPR is applied/has the potential for application in many areas of society involving biology. What are some examples?
e.g., biofuels, food
industry, plant biotechnology, livestock, drug discovery, control of
infectious disease, treatment of genetic diseases like Sickle Cell disease - which CRISPR has been used as a therapy for
b.
(Example of a question that is very detailed and will not be asked)
What does the abbreviation PAM, used for CRISPR genome editing, stand for?
(a) Purine-adenine mutation.
(b) Polymerase alpha binding motif.
(c) Plasmid activating motif.
(d) Protospacer adjacent motif
(e) Pathogenic activating mutation.
d
