Introduction to CRISPR

technology
Mayack Lab

Saleh Ghanem
Overview
• How was CRISPR first found?

• Outline of how CRISPR works

• Methods of introducing CRISPR

• Analysis and comparison of previous studies conducted on D. Melanogaster

• Selection processes

• Brief explanation of FACS

How was CRISPR first discovered?

• Observed in bacteria as a part of its immune system, Cas9.

• Adaptive immunity in bacteria and Archaea.

• Enables bacteria to recognize and degrade invading sequences.

Discovery of CRISPR / Mechanism
Discovery of CRISPR / Mechanism
PAM sequence examples:
Discovery of Crispr / Mechanism
Methods of introducing CRISPR
Analysis and comparison of previous studies conducted on
D. Melanogaster
 Selection processes
In Vitro In Vivo

- FACS - Select the desired phenotype

- Single Cell cloning

- Positive and negative selection

…etc
Overview of FACS
Overview of Positive and negative
selection
• Genetic information that has been cut with CRISPR may not always repair like it is

expected to……

• Needs to select against cells that have part or none of the desired effects…

• Antibiotic selection in two phases…

dCas9
Is Crispr really modifying bases?
• Short answer: no.

• One of three things happens when a double stranded break is incurred:

1- DNA gets accurately repaired

2- DNA gets inaccurately repaired and there are some mistakes

3- The DNA is repaired via a template that creates an entirely new sequence.
CRISPR
• Goal of inserting a gene into E.Coli.
• Standard place that a lot of genes are inserted: between ybhH and ybhD genes
• Search for the sequence on :https://www.ncbi.nlm.nih.gov/genome/browse/
CRISPR
After searching for our organism, Click the first link at the top next to
“Reference Genome” then scroll down to the bottom and we should see
this:
CRISPR
• Using the tools icon we can search for the ybhD gene and the gene after that is ybhH, between these two is
where the gene should be inserted.
• Knowing the sequence between the two genes is essential for guide RNA design. (the spacer mentioned earlier)

Few considerations:
- Spacer should be 20 bases
- Should end in a PAM sequence

The next step would be to find NGG (PAM) on the top strand, the 20 nucleotides coming before this would be our
spacer.

In order to select an optimal spacer, this tool is useful:

https://www.atum.bio/eCommerce/cas9/results?multipleContacts=false

Also with regards to off targets, CHOPCHOP can be used to see the number of possible off targets based on the
spacer

https://chopchop.cbu.uib.no/
 CRISPR
Region between ybhH and ybhD:

TTATTACTCCGGAAAATGGAAGCGACGATTTTGGGTGGCTGGCCGTTAAAAATT
TTAACTGCATTTAGCCAACTTAAATTAATGAAAAAATGTTATTAATCGTTGAGCT
AAAGTCATTAGAGATGCTTTGCCCTTAATGTAACCATATCGCAATAAGTTATGTT
TTTAAATTGAGGGCATTATTA

This sequence can be used as the spacer:

ATGGAAGCGACGATTTTGGG
Odin kit: genome engineering class
Rolling circle PCR amplification to insert our spacer.

Forward primer

GTCCTAGGTATAATACTAGT + Spacer + GTTTTAGAGCTAGAAATAGC

Reverse Primer

GCTATTTCTAGCTCTAAAAC + Spacer + ACTAGTATTATACCTAGGAC

Homology arms.****
Important note:

• Conceptually, designing the guide RNA will follow the same rules, however the
method/primers outlined today were specifically tailored around The ODIN Kit.
References *
• https://www.creative-biolabs.com/fluorescent-activated-cell-sorting-facs-technology-for
-single-cell-isolation.html
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5901406/
• https://www.sciencedirect.com/science/article/pii/S1673852713002130