National Postdoc Appreciation Week

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Presentation transcript:

National Postdoc Appreciation Week Hands-on CRISPR/Cas9 Design Workshop Exploring CRISPR/Cas9 Systems with the Biomedical Research Core Facilities Thomas Saunders, Ph.D. National Postdoc Appreciation Week September 19, 2014

Genome Editing: Wild Type Cas9 Ran et al. 2013. Nat Protoc. 8:2281

Genome Editing: Cas9 Nickase Ran et al. 2013. Nat Protoc. 8:2281

Workshop Goals Install Lasergene Software Find Mouse Gene Names Find Genomic Sequence Submit Genomic Sequence for sgRNA Select sgRNA for knockouts/knockins Cas9 Cloning Strategy in pX330/pX458/pX459/pX460 plasmids Order Oligos for Cas9 Plasmids

Download DNAStar Software https://wiki.umms.med.umich.edu/display/UMHSHELPDESK/Medical+School+Applications Or use Search Term “MSIS Medical School Applications” click on "login" kerberos password click on "Medical School Software” scroll down to "Lasergene” & install

A Famous Gene: P53

Find Genomic Sequence

Find Genomic Sequence

Find Gene Name

Find Gene Name

Find Genomic Sequence

Select All Text & Copy

Use Paste Special to Paste Text

Save File as .txt

Save File as .txt

Open Trp53 mus.txt in SeqBuilder

Save as Trp53 mus.sbd File

Identify Critical Exon for Knockout

Identify Critical Exon for Knockout

Identify Critical Exon for Knockout

Identify Critical Exon for Knockout

Find Critical Exon Sequence

Paste Sequence into Online CRISPR Tool

Paste Sequence into Online CRISPR Tool

Paste Sequence into Online CRISPR Tool

Paste Sequence into Online CRISPR Tool

Online CRISPR Tool Exon 2 (83 bp)

Exon 2 Results (83 bp) 2

Exon 2 Results (83 bp)

Online CRISPR Tool Exon 4 (261 bp)

Exon 4 Results (261 bp)

Exon 4 Results (261 bp)

Exon 4 Potential Off Target Hits

Exon 4 sgRNA Target 5’ AGTGAAGCCCTCCGAGTGTC AGG 3’ PAM

Ran, et al. 2013. Nat Protoc. 8:2281-2308.

pX330 Plasmid Addgene.org Plasmid 42230 pX460 Plasmid Cas9 Nickase Ran, et al. 2013. Nat Protoc. 8:2281-2308.

pX459 (GFP) Addgene.org Plasmid 48138 pX458 (puro) Ran, et al. 2013. Nat Protoc. 8:2281-2308.

pX330 Plasmid Addgene.org Plasmid 42230 Ran, et al. 2013. Nat Protoc. 8:2281-2308.

Ran, et al. 2013. Nat Protoc. 8:2281-2308.

Exon 4 sgRNA Target Oligos to Synthesize 5’ AGTGAAGCCCTCCGAGTGTC 3’ 3’ TCACTTCGGGAGGCTCACAG 5’ Oligos to Synthesize 5’ CACCGAGTGAAGCCCTCCGAGTGTC 3’ CTCACTTCGGGAGGCTCACAGCAAA

Clone into pX330 See Ran, et al. 2013. Nat Protoc Clone into pX330 See Ran, et al. 2013. Nat Protoc. 8:2281-2308 for experimental methods Anneal Oligos Ligate into pX330 plasmid digested with Bbs I Transform bacteria Analyze colonies for correct sequence insertion Test Plasmid for activity in vitro or in vivo

Mutation Screening by Analysis of PCR Fragments

Cas9 Targeting in ES Cells

Mutation in Cultured Mouse Eggs ACCACTCCCTATCTTTCTTCTCCCTCCCTGCAGGTATGAATGCTGCCGTCA DNA Example Chromatogram Reveals Mutation: Superimposed Mutant and Wild Type “Peaks-on-Peaks” Sequences.

Mutant Mouse Pups Cel I

Mutation in Mouse Founder ACCACTCCCTATCTTTCTTCTCCCTCCCTGCAGGTATG----CTGCCGTCA ACCACTCCCTATCTTTCTTCTCCCTCCCTGCAGGTATGCTGCCGTCAGGGC Mutant Mouse Sequence – Out of Frame mRNA

Transgenic Core Expertise As new investments are made into the cores, our main goal is just to make sure that the main BRCF audience – U-M faculty, staff, Pis, post-docs, grad students and researchers know about all of the resources and expertise the BRCF have to offer.