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

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

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 https://upload.wikimedia.org/wikipedia/commons/0/08/12_Hegasy_Cas9_Immun_Wiki_E_CCBYSA.png

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.” https://upload.wikimedia.org/wikipedia/commons/1/1c/13_Hegasy_CRISPR_pre_crRNA_Wiki_E_CCBYSA.png

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 https://www.synthego.com/guide/how-to-use-crispr/pam-sequence

A short history… Year Advancement 1987 . 2005 . . 2012 . 2015 1987 . 2005 . . 2012 . 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

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).” https://www.synthego.com/guide/how-to-use-crispr/sgrna https://upload.wikimedia.org/wikipedia/commons/f/f4/15_Hegasy_Cas9_DNA_Tool_Wiki_E_CCBYSA.png

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

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

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) * https://en.wikipedia.org/wiki/CRISPR ** https://www.vox.com/2018/7/23/17594864/crispr-cas9-gene-editing

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 https://en.wikipedia.org/wiki/File:16_Hegasy_DNA_Rep_Wiki_E_CCBYSA.png CRISPR 101 e-book Snythego

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.

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 https://en.wikipedia.org/wiki/Off-target_genome_editing 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: https://phys.org/news/2019-04-method-off-target-effects-crispr.html Sept ‘18: https://www.the-scientist.com/news-opinion/new-technique-limits-crispr-cas9-off-target-mutations-64784 DISCOVER-Seq for detection: https://www.genengnews.com/news/discover-seq-detects-off-target-crispr-effects/ See CRISPR folder for articles

Recently… Human Nature First time in HIV patient Documentary on CRISPR directed by Adam Bolt Released March 2019 https://en.wikipedia.org/wiki/Human_Nature_(2019_film) First time in HIV patient https://www.cnn.com/2019/09/13/health/china-crispr-hiv-intl-hnk/index.html Thanks, Bebe!

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