Genome Engineering with Crispr/ Cas9 Workshop

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GENOME ENGINEERING WITH CRISPR/ CAS9 WORKSHOP

Sandra Porter, Digital World Biology Thomas Tubon, Madison College

Bio-Link Fellows, UC Berkeley CKC Campus Wednesday June 8, 2016 DUE 1501553 Building 14, Room 203 Overview

I. What is Genome Engineering?

II. Molecule World – CRISPR/Cas9 Molecular modeling

III. Applications of CRISPR/Cas9 for Genome Engineering

IV. Genome Engineering in the Biotech Classroom

What is Genome Engineering

“…the process of making targeted modifications of the genome, its contexts (e.g. epigenetic marks), or its outputs (e.g. transcripts).

The ability to do so easily and efficiently in eukaryotes especially mammalian cells holds immense promise to transform basic science, biotechnology, and medicine…”

(Hsu, Lander, & Zhang 2014 Cell) Genome Engineering

Applications:

• Recapitulate genetic variants associated with altered biological function in animal or cellular models.

• Manipulate genes to generate synthetic materials.

• Precision engineering of food crops, and possible sources for biofuels (algae/corn).

• Direct correction of genetic defects in vivo of somatic tissue for therapeutics

(Hsu, Lander, & Zhang 2014 Cell) Genomic Engineering Tools

I. Meganucleases (2006): derived from microbial mobile genetic elements

II. Zing Finger Nucleases (2005): design based on eukaryotic transcription factors.

III. Transcription Activator-Like Effectors (TALEs; 2010): derived from Xanthomonas bacteria

IV. CRISPR/Cas9 (2013): RNA-guided DNA endonuclease (Cas9) from the type II bacterial adaptive immune system CRISPR. (Jennifer Doudna/Emmanuelle Charpentier, Feng Zhang, George Church).

“Programmable nuclease-based genome editing”

Comparison of Genome Editing Technologies

System TALEN ZFN CRISPR/Cas9 Target Genome-wide Genome-wide Genome-wide FokI sites FokI sites Requires Requires Repeats Protospacer Adjacent Motif (PAM) Type of Break Double stranded Double or single Double or single stranded stranded

Cost of High High Low (requires only Production ($1000s) ($1000s) plasmid and backbone) (<$100) Construction Difficult, must be Difficult, must be Easy and In- sent to company sent to company house Comparison of Genomic Editing Technologies

Gasiunas, Giedrius, and Virginijus Siksnys. "RNA-dependent DNA endonuclease Cas9 of the CRISPR system: Holy Grail of genome editing?." Trends in microbiology (2013). Clustered Regularly Interspaced Short Palindormic Repeats (CRISPR/ CRISPR Associated Proteins (Cas9) Timeline for CRiSPR/Cas9 Genome Editing Genome Engineering: Clustered Regularly Interspaced Short Palindormic Repeats (CRISPR/ CRISPR Associated Proteins (Cas9) Genome Engineering: CRISPER/Cas9 nuclease-based genome editing

• System has been reduced to a two-plasmid system • One plasmid encodes the human codon optimized Cas9 protein • Second plasmid encodes the target site as well as chiRNA necessary for recognition • Next generation CRISPR systems combine these two coding systems into one plasmid.

Di Carlo et al. 2013 Genome Engineering: CRISPER/Cas9 nuclease-based genome editing

• Nuclease is targeted by guide RNA gRNA that hybridizes to a 20 nucleotide sequence and is terminated by a Protospacer Adjacent Motif (PAM) • Generally -NGG, which occurs every ~32 base pairs in the human genome • Double strand break is created 3 bp upstream of PAM site • Cas9 is non-cytotoxic in hPSCs so can be expressed at higher levels Di Carlo et al. 2013 Double Stranded Break

Non-Homologous End Joining Homology Directed Repair (NHEJ) (HDR)

http://www.ltk.unizh.ch/de/dyn_output.html?content.void=2301&8f0c69bfb3bd9c73e63efd1dac653293 Summary: Genome Engineering

Impact: CRISPR/Cas9 Programmable Biology, Biotechnology, & Medicine Nuclease-based genome editing technology

(Hsu, Lander, & Zhang 2014 Cell) (http://www.tuftssyntheticbiology.com/images/crispr-cas.png) II. Molecular World Biology: CRISPR/Cas9 Molecular Modeling (Sandy Porter)

III. Applications for Genome Editing Genome Therapies First proposed in 1972 with the first trials occurring in 1990.

As of Today, 4541 gene therapies are in clinical phase trials; 1646 emphasize genomic approaches, and 3 involve specific genomic editing tools (www.clinicaltrials.gov).

Success in treating diseases such as multiple myeloma, hemophilia, leukemia, and Parkinson's

Genome Therapies - Concept

Robinton, D.A & Daley, G.Q. (2012) The promise of induced pluripotent stem cells In research and therapy. Nature 481. 295-305 Application I: CRISPR/Cas9 Targeted Gene Editing

Mice with a dominant mutation in the Crygc gene, which causes cataracts could be rescued using By coinjection into zygotes of CRISPR/Cas9, gRNA, and an a wild-type donor ‘repair’ template.

Cell Stem Cell, December 2013 Application I: CRISPR/Cas9 Targeted Gene Editing

This genetic repair of the original dominant Crycg gene is heritable.

Cell Stem Cell, December 2013 Application II: CRISPR/Cas9 Targeted Gene Editing in Humans

(Protein & Cell, April 2015) Application II: CRISPR/Cas9 Targeted Gene Editing in Humans

ARTICLE SUMMARY •First demonstration of targeted genomic modification in a human pre-pre- implantation embryos

•3 Pronuclear model (3PN): polyspermic zygotes that fail to develop normally.

•Targeted repair of the β-globin gene mutation that leads to β-thalassemia

•Whole exome sequencing employed to identify off-site targets.

(Protein & Cell, April 2015)

Engineered CRISPR Solutions Double Nicking

• Better specificity than previous CRISPR/Cas9 methods • Reduce off-target activity by 50- to 1,500-fold in cell lines without sacrificing on-target cleavage efficiency.

Ran et al. Cell 2013 Engineered CRISPR Solutions Double Nicking Transcriptional Activation

Ran et al. Cell 2013 Mali et al. Nature Biotech 2013 Engineered CRISPR Solutions Double Nicking Transcriptional Activation

CRISPRi

Ran et al. Cell 2013 Mali et al. Nature Biotech 2013 Qi et al. Cell 2013 Engineered CRISPR Solutions Double Nicking Transcriptional Activation

CRISPRi HT genetic screening with gRNA Libraries

Ran et al. Cell 2013 Mali et al. Nature Biotech 2013 Qi et al. Cell 2013 Shalem et al. Science 2013 IV. Genome Editing in the Classroom CRISPR activities for enhanced classroom learning Computer-based approaches: •Molecular Modeling of CRISPR/Cas9 (Sandy Porter)

•Data mining, bioinformatics, and design of specific gene-targeted guide RNAs (Tom Tubon)

Cell Culture-based approaches: •CRISPR Gene knockdown of Red Fluorescent Protein in prokaryotes (E. coli) (George Cachaines)

•DIY CRISPR Kits: Biohacker assembled $130-$160 kits to alter genomes of bacteria and yeast for genes that affect easily visualized characteristics (color, smell; Josiah Zayner).

Molecular Biology Laboratory: • CRISPR modification of human cells (Human Embryonic Kidney Cells – HEK293; PSCs – IPS-IMR90-4) and detection of INDELS by T7 Surveyor endonuclease activity. (Coming to an NSF workshop near you! – T Tubon / K. Saha)

Policy, Patents, Commercialization, and Bioethics •”CRISPR-edited mushroom dodges USDA regulations (April 2016): CRISPR edited polyphenol oxidase (PPO). CRISPR activities for enhanced classroom learning Computer-based approaches: •Molecular Modeling of CRISPR/Cas9 (Sandy Porter)

•Data mining, bioinformatics, and design of specific gene-targeted guide RNAs (Tom Tubon)

Cell Culture-based approaches: •CRISPR Gene knockdown of Red Fluorescent Protein in prokaryotes (E. coli) (George Cachaines)

•DIY CRISPR Kits: Biohacker assembled $130-$160 kits to alter genomes of bacteria and yeast for genes that affect easily visualized characteristics (color, smell; Josiah Zayner).

Policy, Patents, Commercialization, and Bioethics •”CRISPR-edited mushroom dodges USDA regulations (April 2016): CRISPR edited polyphenol oxidase (PPO). •CRISPR Patents: Who owns CRISPR Technology? CRISPR activities for enhanced classroom learning

Policy, Patents, Commercialization, and Bioethics •CRISPR-Y CRITTERS http://www.ipscell.com/2015/05/crispr-y-critters/

CRISPR GFP worms

CRISPR-Edited CRISPR-edited pigs CRISPR-edited monkeys Rabbits (park2/pink1) (Dystrophin) (tyrosinase) CRISPR activities for enhanced classroom learning

Author: Jeff Wheelwright, June 2016 Discovery Magazine CRISPR activities for enhanced classroom learning

CRISPR gene editing in prokaryotes: George Cachianes

Introduced CRISPR/Cas9 into his Biotech Classroom at Lincoln High. Students used genetically modified E. coli that expresses Red Fluorescent Protein (RFP). Students use the CRISPR/Cas9 system to target the RFP gene and return the bacteria to their normal color. (IGEM)

CRISPR activities for enhanced classroom learning DIY CRISPR Genomic Editing: Josiah Zayner Indiegogo: $71,271 in crowdsource funding raised. https://www.indiegogo.com/projects/diy-crispr-kits-learn-modern-science-by-doing#/

“Yeast are a commonly used organism in Synthetic Biology because they are one of the simplest Eukaryotes (their cells are similar to mammals like Humans!). Normally, yeast grow with a nice creamy white color. This kit makes specific edits to the ADE2 gene using CRISPR. This causes red pigment to accumulate and the yeast to turn red. Yeast require a more complex media to grow so this kit is more expensive. Everything required to perform these experiments is included in the kit.” CRISPR activities for enhanced classroom learning CRISPR Modification in Human Cells (INDEL detection by flourescence)

Human Embryonic Kidney Cells Human PSCs (WA09) Transfection and GFP Knockout Transfection and mCHERRY through CRISPR-targeting Knockout through CRISPR-targeting CRISPR activities for enhanced classroom learning CRISPR Modification in Human Cells (detection by PCR/T7 Surveyor)

T7 Endonuclease I recognizes and cleaves non-perfectly matched DNA, cruciform DNA structures, Holliday structures or junctions, Guschin DY, Waite AJ, Katibah GE, Miller JC, Holmes MC, and heteroduplex DNA Rebar EJ. A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol. 2010;649:247–256. Review

I. Genome Engineering Technoologies

II. Molecular World Biology – CRISPR/Cas9 Molecular modeling

III. Applications of CRISPR/Cas9 for Genome Engineering

IV. Genome Engineering in the Biotech Classroom

Acknowledgements

Kris Saha Jishnu Saha Tom Tubon Ben Steyer Ty Harkness Madelyn Goedland Anita Bhattacharrya Travis Cordie Henry Hu Dustie Held Tyler Klann Kevin Ortiz Erich Berndt Ryan Prestil Ian Linsmeier Mike Musser Matt Doers

Randy Ashton

Lisa Seidman Jeanette Mowery Mary Ellen Kraus Emily Sanders Sandra Docter Advanced Technological Education DUE 1104210/1501553 Dr. Feng Zhang, Dr. George Church, Boston, MA Boston, MA Dr. Emmanuel Charpentier Dr. Jennifer Doudna Basel, Switzerland Berkeley, California

Precision Genomic Editing:

Cas9/CRISPR guide RNA Design

Design your own gRNA

1) Locate the area of your gene of interest in UCSC Human Genome Browser

• Transgenes should be found in NCBI primary assembly viewer

• Obtain FASTA sequence Design your own gRNA

1) Locate the area of your gene of interest in UCSC Human Genome Browser

• Transgenes should be found in NCBI primary assembly viewer

• Obtain FASTA sequence 2) Go to crispr.mit.edu and input sequence (up to 250 bp, larger sequences must be divided and input individually) Design your own gRNA

1) Locate the area of your gene of interest in UCSD Human Genome Browser

• Transgenes should be found in NCBI primary assembly viewer

• Obtain FASTA sequence 2) Go to crispr.mit.edu and input sequence (up to 250 bp, larger sequences must be divided and input individually) 3) Choose guides that are close to the target site residues and also have a high score, which correlates to a lower predicted chance of off-target effects. Design your own gRNA

1) Locate the area of your gene of interest in UCSD Human Genome Browser

• Transgenes should be found in NCBI primary assembly viewer

1.) Order overlapping primers with target and reverse complement

Addgene.org/static/cms/files/hCRISPR_gRNA_Synthesis.pdf gRNA Construction

1.) Order overlapping primers with target and reverse complement

2.) PCR two oligos together

Addgene.org/static/cms/files/hCRISPR_gRNA_Synthesis.pdf gRNA Construction

1.) Order overlapping primers 3.) Digest backbone vector with target and reverse (Addgene #41824) complement

2.) PCR two oligos together

Addgene.org/static/cms/files/hCRISPR_gRNA_Synthesis.pdf gRNA Construction

1.) Order overlapping primers 3.) Digest backbone vector with target and reverse (Addgene #41824) complement

2.) PCR two oligos together 4.) Ligate together using Gibson Assembly

Addgene.org/static/cms/files/hCRISPR_gRNA_Synthesis.pdf gRNA Design Tools

http://www.addgene.org/crispr/reference/#gRNA gRNA Design Tools

http://www.addgene.org/crispr/reference/#gRNA