CRISPR presentation extended Mouse Modeling
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Transcript of CRISPR presentation extended Mouse Modeling
Why genome engineer?
• Deletions, insertions or modifications of endogenous sequence allow elucidation of gene function / regulatory purposes
• Simultaneous modification of multiple areas can clarify the relation between elements
What qualities are we looking for in gene targeting technology?
It needs to be: • Precise• Fast• Applicable to any strain/species• Inexpensive
Traditional gene targeting• ES Cell work: – Precise modifications– Time consuming– (Typically) limited to a single mutation at a time– Limited strains (and species) – Expensive
Nuclease-based gene targeting• Creation of double strand
break (DSB) followed by…
1. Nonhomologous End-Joining (NHEJ) leading to small insertions or deletions (indels): KO if not in-frame
2. Homology Directed Repair (HDR) in the presence of an exogenous template
(modified from Hsu et al .2014)
Nuclease-based gene targeting
• Meganucleases• ZFN: Zinc-finger + nuclease• TALEN: Transcription activator-like effector +
nuclease• CRISPR/Cas9: Clustered regularly interspersed
palindromic repeat sgRNA + Cas9 nuclease
Zinc Finger Nucleases
- Based on triplet subunit interaction with DNA
- Uses two 9-18 bp recognition sequences with ~5 bp between
Zinc Finger Nucleases• Pros:
– Use of heterodimers w/ FokI causes good specificity/efficiency– KO leads to large deletions; KI also possible– Readily available online– Applicable in many species– Well established: phase II clinical trials
• Cons: – Extreme difficulty making yourself: don’t try– Triplet-based nucleotide-protein interaction causes potentially serious
specificity issues– Expensive and time consuming to make yourself/have made for you
TALENs
(http://geneeditingservice.com/Home.php)
- Uses two 18-20 bp recognition sequences with ~18 bp between-
TALENs• Pros:
– Evolution of the ZFN principle – Protein “code” for base pairing relatively straightforward– Use of this protein w/ FokI causes better specificity/efficiency– KO leads to large deletions; KI also possible– Applicable in many species – Readily available online but able to be made in lab
• Cons: – Relatively expensive and time consuming to make yourself/have made
for you– Nucleotide-protein interaction causes potential specificity issues
(though less seriously than with ZFN)
CRISPR/Cas9• Abridged history:
– 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
– 2005-2007: Several groups note that the sequences between the repetitive elements are consistent with phage sequences; their presence in >50% of bacteria lead to speculation about phage immunity
– 2007-2009: Danisco A/S recognize/utilize CRISPR to improve yogurt/cheese bacterial culture resistance to viruses (Dupont acquire Danisco in 2011 for 6.3 billion)
– 2008: Carte et al. & Marraffini et al. elucidate a role for Cas proteins in the CRISPR phage targeting system
– 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
A bacterial immune system:• Found in ~85% of archaea, 50% of bacteria
• 3 types of CRISPR systems: type II most used(modified from NEB CRISPR webpage)
Repurposing an immune system for genome engineering: minimal type II CRISPR components:
• 3 simple ingredients: – Cas9 protein– crRNA– tracrRNA
• In the target:– Homology to crRNA “spacer” sequence– PAM sequence: NGG for S. pyogenes
(modified from NEB CRISPR webpage)
CRISPR basics: crRNA + tracrRNA = sgRNA
Repurposing an immune system: minimal type II CRISPR components for Gene Targeting:
• 2 simple ingredients: – Cas protein: universal– crRNA– tracrRNA– sgRNA: unique
• Homology to sgRNA “guide” sequence• PAM sequence: NGG for S. pyogenes
(modified from NEB CRISPR webpage)
CRISPR basics: the sgRNA
• ~124 nucleotides total • 20 bp guide:
• 8-12 PAM-proximal nucleotides are critical: “seed sequence”
• PAM not included in sgRNA• Essential:
• Guide:Target• Repeat:Anti-Repeat duplex• Stem loop 1
• Beneficial: • Stem loops 2 and 3
CRISPR basics: the Cas9 protein
• ~4.3 kb• REC lobe: – Rec1 domain– Rec2 domain
• NUC lobe:– HNH domain– RuvC domain– PAM-interacting (PI) domain
CRISPR basics: gene editing in mammalian cells
CRISPR basics: multiple KO in mice
CRISPR basics: specific edits in mice
CRISPR in Mice: specific editing
CRISPR/Cas9• Pros: – Seemingly excellent ability to target any genomic region– All mutations possible short of very large inserts or deletions– Fast– Components available online– Many software programs to help design guide sequences– Utilized in many different species– Cheap
• Cons: – PAM requirement: not too limiting, every 12 bp– Off target effects: some controversy – Mosaic effects
(Jinek et al. 2012)
Into the Wild
Frederick Church – The Andes of Ecuador (1855)
What do you need to do?
• Identify the mutation you want to make:– KO, KI, deletion, etc.
• Identify your strain of interest• Plan your screen• Submit your application (~3-4 months)• Design your components • Prepare your components• Prepare your screen
CRISPR in practice: an overview
FIRST:make sure your mouse isn’t on the shelf!
• Findmice.org
What’s possible with CRISPR?
Indels for knockout via NHEJ: 80-90%• Inserts/Deletions• Cas9 mRNA + sgRNA (+ sgRNA?)• Very straightforward with CRISPR:
• Frameshift mutations not guaranteed to KO (modified from Ran et al. 2013)
“Belt and Braces” aka “double assurance”
• sgRNAs are cheap • Multiple can be injected at once• Testing them is costly
• No risk in double assurance, other than double OT effect
Large deletions via NHEJ: ~30%
• Theoretically: use 2 sgRNA on opposite sides of the sequence to be deleted (or inverted)
• 10 kb reported:
• Yang et al. 2014, 30% for 700 bp deletion… • Worth a try, larger than 10 kb should be done in
ES cells
Point mutation, small tag, lox site KI via HR: • 50-80%, 30-50%, 10-20% targeting efficiency respectively• Cas9 mRNA, sgRNA + donor template• Donor template is 200mer ssODN(IVT)• 20 bp targeted sequence/DSB should be as close to the
edited sequence as possible – 30 bp away reported
• Larger arms of homology typically better: 40-60 bp of isogenic sequence
(Shao et al. 2014)
Large insertions via HR: 10-20%
• Cas9 mRNA, sgRNA and donor template• Donor template is circular plasmid (linear ok)– Prevents integration– Less apoptotic effect
• 0.5-5 kb of isogenic homology on each side of insert (3-5 kb most common)
• No consensus yet on optimal conditions
(modified from Ran et al. 2013)
The Importance of Isogenicity• High fidelity between guide/donor sequence and the recipient
strain genome maximizes the chance of successful targeting
A crucial point for insertions• Repair template should contain a silent mutations
in the PAM and/or seed sequence. Otherwise: – After initial HDR a second DSB/indel can occur – Cas9 cleavage ability can be drained by the donor pool
(Yang et al. 2014)
Strain-dependent superovulation response
Strain-dependent superovulation response
Why is strain important?• Different strains are better suited to different
projects: – B6C3F2 vs. FVB vs. NSG
• Strains react differently to superovulation• Embryos from different strains show varying
survival rates post injection• Chosen strain will affect your sgRNA/template
DNA design: isogenicity is key
Overcoming strain-specific issues with IVF
• CRISPR broadly applicable to many different strains• Strains like NSG have terrible egg collection rates or
return: poor breeding capacity• IVF can overcome this challenge at the expense of
Audra’s sleep– IVF occurs ~12:00 am– Eggs ready to microinject ~8:00 am the following day– Reserved for rare circumstances
sgRNA design tools:
To identify the 20 bp guide sequence
• http://crispr.mit.edu/ • http://zifit.partners.org/ZiFiT/• https://chopchop.rc.fas.harvard.edu/ • http://www.e-crisp.org/E-CRISP/index.html
All of these likely/explicitly based on the C57BL/6J genome
Screening strategy
• Surveyor assay:
• Addition/deletion of restriction sites … no true insight into the mutation
• Use the above methods to screen for mutations followed by sequencing. – Complicated by mosaicism
Mosacism in two respects…
• In terms of WT vs. mutant alleles in an animal…
Mosacism in two respects…
• …and within the pool of modified genes
Screening strategy
• Sanger sequencing: – Problematic
• Clone into plasmids: Sanger sequence clones
• Deep sequencing: another possibility
• Specific insertions: – Internal-external primer
PCR
Making sgRNA/Cas9 templates for IVT
After identification of target site: Many options… an example:• Clone sgRNA target into a plasmid– pX260 or pX330 (with Cas9)– Transform into cells, maxiprep to prepare template
Amplification of templates:
• Use plasmid template in 2 PCR reactions:– One with primers to amplify sgRNA template
– The other with primers to amplify Cas9 template
– High fidelity PCR systems a must
(Harms et al. 2014)
(Harms et al. 2014)
In vitro transcription/clean up
• mMessage mMachine T7 ULTRA • For Cas9 mRNA be sure that the protein is
capped and polyadenylated
• Purify resulting RNA with MegaClear kit, NucAway or phenol-chloroform extraction
RNA quantification
• Nanodrop readings unreliable• Qubit much better: – Fluorescence-based– More sensitive readings– Available in shared equipment
Final concentrations: no consensus
(Singh et al. 2014)
Final concentrations: no consensus
(Singh et al. 2014)
Final concentrations: no consensus
(Yang et al. 2014)
Michael Wiles lab: Peter Kutny: Wenning Qin’s lab:
Cytoplasmic: 100 ng/uL Cas950 ng/uL sgRNA
Pronuclear + oligo60 ng/uL Cas930 ng/uL sgRNAs1 ng/uL 200mer
100 ng/uL Cas950 ng/uL sgRNA120 ng/uL dsODN plasmid
Pronuclear (+C)100 ng/uL Cas950 ng/uL sgRNA20 ng/uL dsODN
Cytoplasmic: 60 ng/uL Cas915 ng/uL sgRNA115 ng/uL sgRNA2
Pronuclear + sc plasmid60 ng/uL Cas930 ng/uL sgRNAs1-20 ng/uL plasmid
Pronuclear + lin plasmid60 ng/uL Cas930 ng/uL sgRNA1-2 ng/uL linearlized plasmid
Pronuclear nickase100 ng/uL Cas950 ng/uL sgRNA150 ng/uL sgRNA220 ng/uL dsODN
VAI Superovulation Strategy
• B6C3F2 embryos– C57BL/6J x C3H hybrid– ~28 eggs recovered/mouse– 85% fertilization rate
• Day 1: inject 5 IU PMS via IP injection into donor B6C3F1 females at 1:00 pm
• Day 3: inject 5 IU HCG via IP injection into donor B6C3F1 females at 11:00 am; mate immediately
• Mate B6D2F1 females to vasectomized males for pseudopregnant mothers at 3:00 pm (no hormones)
• Day 4: microinjection day ~6:00 am– Check donor pseudopreg plugs– Sac donors, remove oviducts and isolate eggs
Nature Protocols 2, 1206 - 1215 (2007) Published online: 10 May 2007
– Isolate fraction of fertilized eggs if necessary: 9:00 am
http://card.medic.kumamoto-u.ac.jp/card/english/sigen/manual/mouseivf.html
Egg removal
Meanwhile in your lab: preparation of injection mix
• Morning of day 4: mix CRISPR components in their proper ratios: – 100 ng/uL Cas9, 50 ng/uL sgRNA, 100 ng/uL template (cyt)– 10 ng/uL Cas9, 5 ng/uL sgRNA, 10 ng/uL template (pron)
– Take an aliquot to run on a gel: verify size and lack of degradation• Run on a denaturing gel (e.g. formaldehyde)
• Hand off to Gene Targeting core ~9:00 am– Centrifuge 10 minutes @ 16,000 x g, 4°C– Pass through Millipore Ultrafree-MC centrifugal filters
2091: Transgenic procedure room
2091: Transgenic procedure room
2091: Transgenic procedure room
• 200°C for two hours• Glass capillary tubes • Slides• Coverslips• Holding pipettes
Eggs loaded into injection chamber
(U of M transgenic core )
2091: Transgenic procedure room
2091: Transgenic procedure room
• …plus filament and capillaries
2091: Transgenic procedure room
(Lobo et al. 2006)
Injections: cytoplasmic vs. pronuclear
http://www.med.upenn.edu/cores/transgenic_and_chimeric_mouse_facility.shtml
The result
(Behringer R., Gertsenstein M., Nagy K. V. and Nagy A., 2014, Manipulating the mouse embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 255)
Healthy
Surviving embryos transferred to pseudopregnant females
http://card.medic.kumamoto-u.ac.jp/card/english/sigen/manual/transfer.html
• Run out the remaining RNA on a denaturing gel?
4 weeks later:
5 weeks after that:
As compared with the earlier mosaics…
CRISPR off-target
• http://mortlockcrispr.blogspot.com/– “In my experience most mouse CRISPR targets have several-to-
many 3-base mismatches, and I’m guessing that most targets will have many 4-base mismatches in mammalian genomes”
– Literature review of off-target mutations (WT Cas9)• 10 papers sought to quantify off-target effects
– How to define/identify an off-target sequence?• Deviation of how many bases? • In the seed sequence (8-12 3’ bp) or the entire protospacer? • Based on scoring analysis by online software?• Concentrations of components• Differences in the sensitivities of off-target assays
CRISPR off-target• 5/10 (50%) were able to detect some degree of off-target mutation.
• But from ~175 OT’s screened, mutations in only 13 (7%) were detected. Several of these OTs had fewer than 3 mismatches to the target. (so 3+ mismatches have a small likelihood of targeting)
• “In conclusion, the consensus from many studies of CRISPR-mediated mouse engineering demonstrates that native Cas9 has a low rate of off-target effects in mouse zygotes. Of course, targets should still be pre-screened when possible to avoid those that will have more potential off-targets, particularly those with fewer than 3 mismatches within the protospacer.”
• ↑ RNA concentration = ↑ mutation frequency = ↑ OT effect
CRISPR off-target (human cells)
CRISPR off-target
CRISPR off-target
• What about traditional ES cell based targeting?• And what about backcrossing?
tru-gRNA
• Truncation of guide sequence to from 20 bp to 17-18 bp (with G(G))
tru-gRNA
tru-gRNA
tru-gRNA
tru-gRNA
• Truncation of guide sequence from 20 bp to 17-18 bp (plus G or GG)
Nickase strategies
• Cas9n (Cas9 D10A) nicks complementary strand
• sgRNA pairs must create 5’ overhangs
Nickase strategies
• Offset between the target sequences should be between -8 and 20 bp.
Refinements:• Addition of RNAsin by JAX @ 0.2 U/uL• Addition of compounds to increase HDR/inhibit NHEJ– SCR7 to inhibit Ligase IV
Refinements: further simplification?
• Why hasn’t this taken over? – Mosacism
Refinements: Cas9 protein
• Reduced off-target effect in human cells
• In principle, less mosacism in mouse zygotes
• Incubate with sgRNA @ 37° C for ~5 min to form a complex prior to injection
Refinements: additional Cas9s?
• Many different type II Cas9 systems left to explore
Refinements: Cas9 KI mouse
Refinements: Cas9 KI mouse
Acknowledgements
VAI vivariumAudra GuikemaTina MeringaBryn EaglesonAshlee Verbis Neil BrandowMat RackhamVivarium Staff
VAIDr. KwangHo LeeDr. Lorenzo SempereDr. Bart WilliamsCassie Diegel
JAXMichael WilesWenning Qin
U of M GT CoreDr. Thom SaundersElizabeth Hughes
The Mice