Reverse Engineering Cells with CRISPR/Cas9 Genome-wide Screens

Question 1

Define

Co-dependent function

Answer 1

• Rescue, AKA resistance/masking/suppressors
• A mutation or defect in one gene is compensated for or alleviated by a second mutation in a different gene.

Question 2

Define

Redundant function

Answer 2

• Synthetic sickness/lethality AKA sensitivity/enhancers/conditional essentiality
• Multiple genes or genetic elements perform overlapping or similar functions within a biological pathway or process.

Question 3

Describe

The cell death - optimal growth spectrum

Answer 3

Synergy = interaction = redundancy = antagonism

Relationship between cellular processes associated with growth and those associated with cell death or apoptosis.

Question 4

Describe

The ways to make a gene knock out/down in mammalian cells?

Answer 4

• RNA interference (knock down, low efficiency, many off-target effects)
• TALENs (engineered restriction enzymes)
• Gene-trapping
• CRISPR/Cas9
• Drug/compound

Question 5

Define

CRISPR/Cas9

Answer 5

• Clustered Regularly Interspaced Short Palindromic Repeats
• Taken from a bacterial adaptive immune system

Question 6

Define

Cas9

Answer 6

• CRISPR-associated protein 9
• Generates DNA DS break, guided by a 20-bp sgRNA 3 bases before PAM

Question 7

What are the two strategies for large-scale gene KO phenotype screening?

Answer 7

• Arrayed
• Pooled library

Question 8

What are the advantages and disadvantages of arrayed screening?

Answer 8

• More possible readouts and easy follow-up experiments
• Slow, expensive
• Requires robots
• Reliance on technical replicates

Question 8

What are the advantages and disadvantages of pooled screening?

Answer 8

• Many readouts from one experiment; built-in biological replicates; can be run by a single biologist
• Fewer possible readouts, requires next-gen methods, need to maintain representation (enough cells)

Question 9

What are the components of library design?

Answer 9

• Choose target regions (all coding regions)
• Generate all potential sgRNAs
• Choose 7-10 sgRNAs per gene (biological replicates)
• Minimize off-target effects
• Optimize on-target KO efficiency

Question 10

How to optimize on-target KO efficiency?

Answer 10

• Optimize sequence (PWM or metling T)
• Avoid alternatively spliced exons
• Avoid the 3’ end where frameshifts could produce proteins
• Favor the coding “+” strand

PWM: By comparing a Position Weight Matrix with a DNA sequence, you can predict potential binding sites for transcription factors or other DNA-binding proteins

Question 11

What does RANKS stand for?

Answer 11

• Robust Analytics and Normalization for Knockout Screens

Question 12

What is the cell essentialome structured like?

Answer 12

• Structured like an onion
• Universal essential genes at the core of essential proteins complexes, as a dense cluster

Question 13

What can chemogenomics reveal?

Answer 13

• Mapping pathways and gene functions
• Mechanisms of action/resistance
• Predict drug-drug synergism/antagonism
• Tumor-specific vulnerabilities

Question 14

How are imperfect knockouts useful?

Answer 14

• Frameshifts near the end of the coding region can lead to a viable (hypermorphic?) protein
• In-frame mutations reveal amino acid-level fitness effects
• Heterozygotes can also be generated

Question 15

How does genome-wide screening avoid investigator bias?

Answer 15

It also includes unimportant and unexpressed genes, as well as less-studied important genes