Advanced Genome Engineering Services and Transgenic Model Generation at MSU’s Transgenic and Genome Editing Facility

BackgroundThe Office of the Vice President for Research Graduate Studies

established MSU-TGEF in January 2016 as part of the Global Impact Initiative (http://research.msu.edu/global-impact/) to provide MSU faculty with comprehensive expertise, resources and support in genetic engineering, molecular cloning and delivery of gene editing reagents to experimental systems.

The mission of MSU-TGEF is to support the in-house generation of transgenic organisms, and cells in a variety of species using the latest genome editing technologies such as CRISPR/Cas. MSU-TGEF supports all stages of genome editing projects from start to finish, including project consultation, experimental design, construct generation, validation and molecular screening; delivery of genome editing components to cells and embryos through transfection, electroporation or microinjection. Currently we provide support for mouse and rat animal models, and eukaryotic cell lines. We are also actively applying our knowledge and skills to other systems including plants, fish, insects and fungi. Our consultations are always free so please call us with questions about how to take advantage of genome editing in your experimental system.

Genome Editing with CRISPR/Cas technologyA

B

C

References:1.Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013 Feb 15;339(6121):819-23.2.Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013 May 9;153(4):910-8.3.Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013 Nov;8(11):2281-308.4.Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014 Feb 27;156(5):935-49.

A. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins are components of an adaptive immune system found in bacteria and archea, designed to remove or silence invading sequences such as viral DNA from host genomes. The CRISPR/Cas system has been adapted to become a revolutionary tool for genome editing1,2,3 in multiple species including vertebrates, invertebrates, plants and fungi. In its simplest form, the CRISPR/Cas mechanism involves a RNA-directed nuclease, such as Cas9, being guided to a specific target sequence in the genome by a crRNA-tracrRNA duplex. The Cas9-RNA complex is able to bind to specific DNA sequences, unwind dsDNA and catalyze a double stranded break repair (DSB) at the target sequence. A hybrid RNA molecule of crRNA and tracrRNA, referred to as single guide RNA (gRNA), has been engineered and shown to be functionally equivalent to the duplex. Identifying gRNA sequences with high on-target and off-target activity with predictive algorithms is a key step in using this system for genome engineering. B. The guide crRNA contains a 20bp protospacer sequence which matches the genomic target, the protospacer is followed by a protospacer-adjacent motif (PAM) such as 5’NGG which must be present in the target genome. The crRNA sequence is complementary to the strand that does not carry the PAM and results in a DSB 3bp upstream from the start of the PAM. C. Overall structure of S. pyogenes Cas9-sgRNA-DNA ternary complex and electrostatic surface potential of Cas9.4

Huirong Xie1 , Elena Demireva1, Nate Kauffman2, Richard Neubig3

1OVPRGS, 2Lyman Briggs College, 3Department of Pharmacology & Toxicology at Michigan State University, East Lansing, MI

MSU-TGEF Current Services CRISPR Genome Editing in Animals At present, CRISPR is the most powerful tool to generate knock-out (constitutive or conditional), knock-in, large insertions or deletions, and point mutations to genetically engineer experimental organisms. •Consultation, project and GE strategy design•Virtual design, target sequence verification and gRNA design•Oligo design and synthesis orders •Design and cloning of HDR template donor construct•In vitro or cell line validation of CRISPR/Cas efficiency for specific gRNAs•Zygote microinjection or electroporation with RNA/DNA/Cas9 protein•PCR genotyping strategy and optimization, genotyping and validation of GE clones or offspring

CRISPR Genome Editing in Cell LinesUsing the same approaches as for editing animals, we can transfect CRISPR/Cas components into target eukaryotic cell lines of different linages with additional services, specifically:•Oligo design and cloning for plasmid delivery of CRISPR/Cas •Cell line transfection via lipofection or electroporation Isolation of transfected single cells by FACS (Flow Cytometry Core) •Expansion and screening of clonal cell lines for positive clones

gRNA analysis and selection Construct design and cloning

Delivery of CRISPR/Cas components by Microinjection

By Embryo Electroporation (*Newly developed*)

Generation of modified animals Genotyping and validation of F1(PCR, Seq, Southern)

ProjectsWith Pups

Strategy Live pups

Edited Pups Editing Efficiency

Gene 1 Transgenic Conventional 62 3 4.8%

Gene 2 Knock-out CRISPR 6 5 (3 large deletion) 83.3%

Gene 3 Knock-out CRISPR 12 11 (3 large deletion) 91.6%

Gene 4 Point Mutation CRISPR 6 6 (1 homo, 5 het) 100%

Gene 5 Conditional KO (1 loxp)

CRISPR Oligo 6 5 (1 with 2 alleles) 83.3%

Gene 6 Conditional KO (2 loxp)

CRISPR construct

11 1 het 9%

CRISPR Genome Editing in Animals

Screening and validation of positive clones:- PCR/T7E1 assay, Western, Functional Assays

GFP

GFP

GFP

GFP

Expand clonal lines

Electroporate with Neon nucleofector

FACS sort single GFP+ cells

CRISPR Genome Editing in Cells

Microinjection for conventional transgenic generationWe utilize microinjection to generate models with conventional: pronuclear injection to generate traditional transgenics or 8-cell/blastocyst ESC injection to generate chimeric mice.•Design and cloning of targeting construct•Pronuclear zygote injection of DNA for transgene targeting•Blastocyst/8-cell injection of ESC for homologous recombination•PCR genotyping strategy and optimization, founder/chimera genotyping and F2 transmission testing

Conventional Constructing and ES cell Targeting We can design and clone targeting vector constructs for generating conventional homologous recombination animals or cell lines. We also provide ESC targeting, expansion and screening services. CRISPR will be used to improve ESC targeting efficiency.

Embryo and Sperm Cryopreservation We provide embryo and sperm cryopreservation services for mutant lines and strains of mice (rats, embryo cryopreservation coming soon).

Summary of completed projects and efficiency ratesBased on the projects we have completed in 2016 we find that the efficiency of CRISPR-mediated targeting (Gene 2 - 6) is dramatically higher than that for conventional transgenic targeting (Gene 1).

Contact:Elena Demireva, Ph.D. ; demireva@msu.edu, 917-519-3853Huirong Xie, Ph.D.; xiehuiro@msu.edu, 513-687-6607

Office/Lab: Bio-Engineering Facility, 775 Woodlot Drive, East Lansing, MI 48824

Mouse Recovery We can revive mice from frozen sperm or embryo archives from worldwide resources. We will use IVF and embryo transfer approaches to produce live pups from frozen sperm. Or we will thaw and transfer embryos to bring back live animals from frozen embryos.

Embryo Transfer We also provide embryo transfer services to eliminate viruses from contaminated mice, or speed breeding especially when mice from other facilities are brought to the quarantine facility here.

Nishimasu et al. 2014