1. CRISPR: What is it?
Biotech Ethics, Fall ‘18

2. CRISPR & Cas
CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats
Cas = CRISPR associated proteins
CRISPR/Cas System:
Biological role: adaptive immunity in prokaryotes (bacteria & archeae)
Biotechnology: gene editing, alterations in gene expression
Mechanism varies among different classes and types of CRISPR/Cas systems; CRISPR/Cas9 most well known

3. Adaptive Immunity with CRISPR in Prokaryotes
Prokaryote retains a ‘record’ of the invading organism by clipping a portion of DNA
This spacer sequence is inserted into the genome adjacent to a cas Operon
Cas
Complex of RNA and protein
Some systems have two RNAs: cr and tracr
Protein can be a complex of multiple polypeptides or one large protein
Contains helicase and endonuclease activities
Cas complex cleaves foreign DNA (plasmids, viral DNA) if reinfection occurs

4. CRISPR Gene Locus
Notice the cas Operon and the adjacent array of CRISPR sequences
The crRNA determines the specificity but works together with the tracrRNA

5. A short history… Year Advancement 1987 . 2005 . . 2012 . 2015
2015
CRISPR sequences recognized But function unclear
Proposed role of CRISPR in prokaryotic adaptive immunity. Strong evidence provided a couple of years later
Guide RNA engineered to target any DNA
Cpf1

6. Some key players in the discovery & early development
Jennifer Doudna, Univ CA, Berkeley
Emmanuelle Marie Charpentier, Umea Univ, Sweden (current position at the Faculty of Science of Sorbonne University in Paris, France)
Feng Zhang, Broad Institute, Boston
Patent controversies continue even now

7. Did techniques exist previously for gene editing or silencing?
Recombinant DNA proteins that recognize a specific DNA sequence and cut the DNA
Zinc finger nucleases
TALENs
Both require a custom made protein for each DNA target
siRNA/RNAi
Can be used to turn genes off temporarily, not permanently

8. Why was CRISPR such a breakthrough compared with previous techniques?
Like ZF nucleases and TALENs, CRISPRs irreversibly knock out the gene and its expression, but CRISPRs can
Target multiple sites at the same time by simply adding different gRNAs (*e.g. 62 pig genes simultaneously)
gRNAs easier and faster to design
Much less expensive and more rapid (**e.g. <$100 in a few hours vs. $1,000’s over weeks or months) * **

9. Synthetic vs. Naturally Occurring RNA
Bioengineering of the CRISPR/Cas9 system has made it even more efficient, for example, fusing the crRNA and tracrRNA into one unit called Single Guide RNA (sgRNA)

10. What can the CRISPR/Cas systems be used to accomplish?
Deletion/inactivation of genes
Cutting & Homologous Recombination
Site-directed mutagenesis
Gene activation (increased transcription)
Target and degrade RNA vs. DNA
See Video #1
[NHEJ = non-homologous end joining]
[HDR = homology-directed repair]

11. Some Applications of the Technology
In research to accelerate our understanding of gene function within genomes
Development of new antibiotic and antiviral treatments
Gene Editing to eradicate disease
Gene Drive to alter entire species to eliminate unwanted or add desirable traits. See Video #2.

12. A Few Concerns, Problems, & Challenges
Off-target effects in the genome
Causing cancer
Altering a species in an irreversible manner with unforeseen negative consequences
Delivery of the gene therapy

13. See our website for more links and videos