1. CRISPR: What is it? Self & World: Impact of Technological
Advances in Biotechnology, Fall ‘19

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
Endonuclease is non-specific
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
crRNA
17-20 nt long
Complementary to target
tracrRNA
Tethers the crRNA to the Cas nuclease to increase binding
tracr is an abbreviation for trans-activating CRISPR RNA, pronounced “tracer RNA.”

5. How do bacteria avoid cutting their own DNA?
PAM: protospacer adjacent motif
Short specific sequence following the target sequence
Required for cleavage by Cas nuclease
PAM sequence located 2-6 nt down from the target sequence that matches the gRNA
Cas cuts 3-4 nt up from the PAM
Researchers would be limited if only one PAM
Design experiments using different Cas endonucleases
Like RE’s, different bacteria have systems that recognize different PAMs

6. 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 (gRNA) engineered to target any DNA
Cpf1, an alternative to Cas9
Nature Comm (2018) article about improved cpf1

7. Synthetic vs. Naturally Occurring RNA
Bioengineering of the CRISPR/Cas9 system has made it even more efficient, for example, use of a Single Guide RNA (sgRNA)
sgRNA contains both the crRNA and tracrRNA in one unit
Terms Single Guide RNA (sgRNA) and Guide RNA (gRNA) are sometimes used interchangeably, but gRNA is many times reserved for the crRNA separate from the tracrRNA
“While crRNAs and tracrRNAs exist as two separate RNA molecules in nature, sgRNAs have become the most popular format for CRISPR guide RNAs with researchers, so the sgRNA and gRNA terms are often used with the same meaning in the CRISPR community these days. However, some researchers are still using guide RNAs with the crRNA and tracrRNA components separate, which are commonly referred to as 2-piece gRNAs or simply as cr:tracrRNAs (pronounced CRISPR tracer RNAs).”

8. Some key players in the discovery & early development
Jennifer Doudna, Univ CA, Berkeley
Emmanuelle Marie Charpentier, Umea Univ, Sweden (current position: Director at the Max Planck Institute for Infection Biology in Berlin, Germany)
Feng Zhang, Broad Institute, Boston
Patent controversies continue even now

9. 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
TALEN:
TAL = Transcriptional-activator like
TALEN = TAL-effector DNA binding domain
RE engineered to bind to DNA

10. 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) * **

11. 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]
Knock in/out: insertion or deletion of genes
Gene tags: real time visualization of NA by attaching GFP to dCas9 (endonuclease-deactivated) or gRNA
Screening: CRISPR libraries to screen more rapidly for what you want (e.g. drug targets)
Anti-CRISPR: inhibitor of Cas9 from phages, greatly reduces off target effects with minimal decrease in activity
CRISPRi/a: interference (gene silencing)/activation
CRISPR 101 e-book Snythego

12. 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.

13. A Few Concerns, Problems, & Challenges
Off-target effects in the genome
Active area of research
Various ways1 to improve (or detect) such as Anti-CRISPR 2
CRISPR 101 e-book Snythego
Causing cancer
Altering a species in an irreversible manner with unforeseen negative consequences
Delivery of the gene therapy
Detection:
Apr ‘19:
Sept ‘18:
DISCOVER-Seq for detection:
See CRISPR folder for articles

14. Recently… Human Nature First time in HIV patient
Documentary on CRISPR directed by Adam Bolt
Released March 2019
First time in HIV patient
Thanks, Bebe!

15. See our website for more links and videos