Bart Williams, PhD Van Andel Research Center

Transcription

1 A History of Genome Editing in the Laboratory Implications for Translational Applications Bart Williams, PhD Van Andel Research Center Introduction by Matthew Denenberg, MD DeVos Childrens Hospital Disclosures: Bart Williams, PhD has affiliations with Surrozen.

2 Overview of Discussion Historical Perspective of Genetically Engineered Mouse Models Enabling Technologies PCR-based amplification Reduced cost of genomic sequencing CRISPR/Cas9 Background CRISPR/Cas9-mediated genetically engineered rodent models Deletions Point Mutations Larger Insertions Discussion on Future Therapeutic Uses

3 Methods to Create Genetically Modified Mouse Strains Pronuclear Injection of Transgenes Embryonic stem cell based homologous recombination Standard Knockouts Creation of Conditional Alleles

4 Building a Targeting Vector to Manipulate DNA (pre-1998)

5 Building a Targeting Vector to Manipulate DNA (pre-1998)

6 Building a Targeting Vector to Manipulate DNA (pre-1998)

7 Cost of Sequencing DNA

8 Speed of Sequencing DNA In the 1970s, you could get a PhD for sequencing a few hundred bases of single gene The first human genome took over a decade to complete You can now sequence an entire genome in days Some sequencing centers now sequence 250 Billion base pairs every day

9 Planning Genomic Modification Experiments (circa 2018) Access publically available genomic databases online Order primers for PCR (polymerase chain reaction) Run PCR

10 Examples of Impact Robinow Syndrome My laboratory is currently using CRISPR-based technologies to model this in mice and rats

11 Summary of Mouse Embryonic Development Day 0.5 Day 1.5 Day 2.5 Day 3.5 Day 19

12 Design of a Transgene-Containing Vector Clone DNA pieces in a the desired order using restriction enzymes and bacteria. Cyagen Biosciences

13 Methods to Create Genetically Modified Mouse Strains Pronuclear Injection of Transgenes Embryonic stem cell based homologous recombination Standard Knockouts Creation of Conditional Alleles

14 Classical Transgenics First Developed in 1982 Advantages Quickly Create Mice Expressing Genes in Specific Tissues Have fertile founder mice within 10 weeks of injection Disadvantages Insertion of the Transgene is Random (no two founders are the same in copy # or insertion site) Source: Boston University Transgenic Core

15 Derivation of Embryonic Stem Cells

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17 Summary of Mouse Knockout Process (Capecchi Approach)

18 Mouse Knockouts ES cell-mediated Germline Modification Advantages Can control exactly where the mutation is Can make very discreet changes (point mutations) Disadvantages (relative to normal transgenics) More time consuming and expensive Germline (global) knockouts are often embryonic lethal

19 Cre-Lox System Overcome embryonic lethality or restrict gene alteration to specific tissues Based on a bacterial DNA recombination system Floxed genes are created by ES cell-mediated techniques

20 Cre-Lox System in Transgenic Mice

21 Time Commitment for Creating Modified Alleles via ES cell modification Build Targeting Vector (several weeks to a few months) Electroporation and Clone Screening (1-3 months) Blastocyst Injection to create fertile chimeras (3 months) months $15,000 to $25,000 and up plus labor Breed to demonstrate germline Transmission (2 months) Cross to FLPeR Mouse and Screening (2 months) Breed to desired cre mouse and screen (2 months)

22 Zinc finger nucleases TALENs CRISPR/Cas Genome Editing All three can be use via pronuclear injection (rats or mice) Can be used on any cell type.

23 CRISPR/Cas9 1987: Ishino et al. notice repeating sequences in bacterial genomes but can t determine their function 2002: CRISPR coined as a term during elucidation of repeat genes Clustered Regularly Interspaced Short Palindromic Repeats Cas= CRISPR-ASsociated : Sequences between the repetitive elements are consistent with phage sequences; their presence in >50% of bacteria lead to speculation about phage immunity

24 CRISPR/Cas : Danisco A/S utilize CRISPR to improve yogurt/cheese bacterial culture resistance to viruses 2012: Charpentier, Doudna & Siksnys groups demonstrate that CRISPR/Cas9 can be used for gene editing in bacteria. 2013: CRISPR/Cas9 shown to work in eukaryotic cells and in genetic engineering of mouse zygotes

25 CRISPR/Cas9 System Results of PubMed search for Cas9 # Total Publications ~3500 (estimated)

26 CRISPR Systems Evolved to Protect Bacteria from Phage Infections

27 Basics of System in Bacteria Three components Needed to Cleave Viral Pathogen DNA Cas9, tracrrna, and crrna attached to spacer (sequence-specific binding) Also somewhat restricted by PAM sequence

28 Basics of System in Mammalian Cells Two components Needed to Cleave DNA Cas9, guide attached to spacer (sequence-specific binding) Also somewhat restricted by PAM sequence

29 Requirements for Genome Editing 1. Cas9 2. sgrna (Guide) 3. Donor DNA Also require some Host RNA processing Factors (RNAse III) that seem to be present in all cells.

30 Ordering CRISPR Reagents Cas9 Protein or mrna tracrrna 62 bp RNA oligo crrna 20 base pairs of complimentary sequence immediately 5 to the PAM sequence on the 5 end of a crrna scaffold Don t have to clone just order RNA oligos and use them as soon as they arrive

31 Creating Transgenics with CRISPR Time commitment: 3 weeks ES cell approaches 15 months Cost: $2,000 - $5,000 ES cell approaches - $25,000

32 Creation of Indels (Insertions and Deletions) Cas9 (mrna or Protein) sgrnas (1 or 2) or tracrrna and crrna separately Target to specific sites you can find in databases

33 CRISPR/Cas9 Proof of Principle: Deletion of Tyrosinase

34 CRISPR/Cas9 Proof of Principle Identified sgrnas to target the Tyrosinase Locus In vitro transcribe Cas9 mrna and sgrnas for microinjection Microinject one embryos that should generate dark colored mice Bart Williams Cassandra Diegel Casey Droscha Mitch McDonald Kevin Maupin Bryn Eagleson Tristan Kempston Audra Guikema

35 CRISPR/Cas9 Optimization 9 of 19 liveborn offspring were white No visually detectable chimerism What used to take months now takes 4 weeks

36 CRISPR/Cas9 Modifications

37 CRISPR Discussion Points Off-target concerns? Can be used on any species where you can inject one cell embryos Don t need an established ES cell line No wet lab work necessary. You can order all the required reagents from companies. You could also outsource the injections to companies.

38 Discussion of the Practicality and Ethics of Transferring this to Human Therapies

39 Discussion of the Practicality and Ethics of Transferring this to Human Therapies