CRISPR/Cas9 Gene Editing

Forward Genetics

Approach to find the genetic basis responsible for a phenotype.

Use sequencing to find gene responsible for mutation

Reverse Genetics

A method to study gene function by specific gene modification and analyzing the phenotypes. 

Create mutation and see if mutation caused the phenotype in the tested organism 

Types of Gain of function experiments

Over expression in cDNA, mRNA, protein or genetic mutation by SNP, insertion, or deletion

Loss of function

RNA interference or mutations via gene editing

Genome Editing to study GOF or LOF: Permanent or transient?

Permanent irreversible

Ectopic Expression to study GOL: Permanent or transient?

Transient or integrated can be reversible

RNAi/shRNA to study LOF: Permanent or transient?

Transient

Target of genome editing?

Non-coding region (98% of the genome)

Target of ectopic expression?

cDNA, has a size limit

Target of RNAi/shRNA?

mRNA or non-coding RNA 

RNA Interference (RNAi)

1. Uses a dicer to cut RNA into smaller fragments

2. The shorter fragments can be loaded into risc complex then the RNA will be degraded so that the RISC complex can silence the RNA

Used to study LOF

What is unique to genome editing?

Clonal populations, screening is needed by serial dilution, each individual cell will have clonal variation

2 repair pathways in genome editing? 

NHEJ & HDR

NHEJ

Creates small deletions or insertions (indels) that can result in frameshift mutations, disrupting gene expression

most common repair pathway, not cell cycle dependent

HDR

Uses a donor template that can introduce precise insertion or replacement

G2/S phase only, low frequency

How are rates of HR increased? 

Synchronize cells, some companies sell HR enhancers to increase the rate of HR by inhibiting NHEJ 

Before CRISPR, what were the gene editing tools used?

• Zinc Finger Nucleases (ZFNs)

• Transcription Activator Like Effector Nucleases (TALENs)

Both fuse a specific DNA binding domain with a DNA cleavage domain, both are not used much anymore because crispr is easier to use

CRISPR stands for

Clustered Regularly Interspaced Short Palindromic Repeats 

What is CRISPR?

A bacterial defense system with repeat sequences every 20 nucleotides (spacer sequences)

• Bacteria transcribe the DNA elements to RNA upon viral infection, the RNA guides a nuclease to the viral DNA to cut it, providing protection against the virus

CRISPR STEPS

1. Virus invades bacterial cell

2. New spacer is derived from virus and integrated into CRISPR sequence

3. CRISPR RNA is formed

4. CRISPR RNA guides molecular machinery to target and destroy viral genome

Parts of the CRISPR/Cas9 Nuclease Complex

• SpCas9 - nuclease 

• 2 RNAs - tracrRNA (loop structure), sgRNA (complement to DNA target (20nt)

• PAM (ngg) - 3’ of DNA target

2 main parts of CRISPR/Cas9

1. Cas9 endonuclease

2. CRISPR RNA (crRNA)

Why is Cas9 the preferred system?

It only requires two components, the other cas’s require more components

Why is it important that Cas9 cleaves the DNA 5’ of PAM?

To ensure that only sequences adjacent to a PAM are recognized and cut 

2 Cas9 variants

1. Cas9 Nickase

2. dCas9

Cas9 Nickase

creates a single DNA strand break instead of a double strand break which is good for less toxicity

dCas9

Will target a specific location but will not break the DNA 

How to design Cas9

Find a region of interest with a PAM, design a guide to limit damage, find the exact location using BLAST

What is important to remember about designing guide RNAs for knock-ins?

Make sure the cutting site is within 10 nucleotides of the target site

CRISPR/Cas9 Applications

1. LOF studies - knock out a gene by frameshift indel, deletion

2. SNP studies - knock in point mutations

3. Insert tag (fluorescent, epitope) - knock in DNA fragment coding epitope or fluorescent tag 

4. Large chromosome deletion, inter chromosome fusion - low frequency events, needs selection marker 

T7 Endonuclease Assay

Can be used to detect indels, when indels are detected the nuclease will cut the indels/mismatched structure can be removed during the denaturing step of PCR

Knock-In Design

Use a template to insert target sequence a silencer may need to be used on either side of the sequence to prevent it from being cut

For use KI use a restriction enzyme to find during sequencing

Electroporation

The most commonly used method to deliver CRISPR with HDR template into KI cells, it has higher efficiency than the other methods

Why is RNP preferred for delivering CRISPR?

RNP has higher transfection efficiency

3 ways to deliver CRISPR

1. Plasmid

2. RNP

3. Viral

Plasmid delivery of CRISPR

Cas9 + gRNA all in one, and a selection marker (GFP)

• transient transfection (electroporation or liposome transfection) 

RNP

Cas9 protein + sgRNA

• transient transfection (electroporation or liposome transfection)

• less off target, lower toxicity

Viral Delivery

Lenti - Cas9 + Lenti-gRNA or Lenti-Cas9 - gRNA all in one

Pro: can infect hard to transfect cells

Con: integrate into the genome, can increase off target cutting

Single Cell Cloning

3 methods:

1. Limited Dilution: plate on 96 well plate at density of <1 cell per well. plate few cells on big plates and pick colonies

2. FACs - sorts a single cell into 96 wells

3. CellRaft Air System

CellRaft

Uses an imaging system to track the growth of clones from single cell and pick the chips that contain single cell clones to plates

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A chamber with removal chips that can be removed once the clones are on the clones are on them, this is good because cells do not like growing independently so they are able to grow together but can be removed one at a time for single cell experiments 

Genotype based clone screening

Genomic PCR screen to edit the region 

1. Run agarose gel to detect size change (deletion or insertion) 

2. design restriction enzyme site for screening 

3. DNA sequencing

Phenotype based CRISPR clone screening

• Measure protein (antibody based assays), western blot, flow, immunoflourescence, ELISA

• Measure RNA (qRT-PCR)

• other phenotypes can be measured via fluorescence, luminescence morphology

2 ways to edit without creating double strand breaks

Prime Editing

Base Editing

Prime Editing

Uses Cas9 nickase to fuse reverse transcriptase and guide RNA, editing template, and binding site into the DNA

Cas9 nickase + Reverse Transcriptase

Base Editing

Fuse Cas9 protein with a base editor to convert the bases in a window of 3-5 bases. There are different editing and activity windows.

gRNA + Editing template (RTT) + Prime binding (PBS)

Prime Editing Steps

1. The prime editing complex binds to the target DNA, Cas9 Nickase nicks one strand of the DNA and generates a flap

2. The prime binding site of the RNA binds to the DNA flap

3. The editing template sequence is reverse transcribed into DNA

4. Target DNA is repaired with the new reverse transcribed DNA

Base Editing Steps

• Cytosine base editors (CBEs) convert C to T

• Adenine base editors (ABEs) convert A to G

• Base editors modifies a 3 to 5 base window 18 bases upstream of the PAM

How can dCas9 be used for genomic targeting?

dCas9 can be used to target promoter or repressor of the sequence

(dCas9 activator/repressor)

• CRISPR interference - dCas9-Repressor

• CRISPR activation - dCas9-Activator

LOF screen using Lenti-CRISPR library

• NextGen Sequencing to count gRNAs, compare gRNAs representation pre and post selection

• gRNAs targeting genes enhancing survival under selection will be dropped out from the population

• gRNAs targeting genes impairing survival will be enriched

Why should MOI be less than one for the lenti-screen?

To ensure most of the cells are infected by no more than one virus