2. 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
3. What qualities are we looking for in
gene targeting technology?
It needs to be:
• Precise
• Fast
• Applicable to any strain/species
• Inexpensive
4. Traditional gene targeting
• ES Cell work:
– Precise modifications
– Time consuming
– (Typically) limited to a single mutation at a time
– Limited strains (and species)
– Expensive
5. 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)
7. Zinc Finger Nucleases
- Based on triplet
subunit interaction
with DNA
- Uses two 9-18 bp
recognition sequences
with ~5 bp between
8. 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
10. 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)
11. 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
12. 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)
13. 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)
22. 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)
24. 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
28. 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)
29. “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
30. 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
31. 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)
32. 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)
33. The Importance of Isogenicity
• High fidelity between guide/donor sequence and the recipient
strain genome maximizes the chance of successful targeting
34. 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)
37. 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
38. 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
39. 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
40. 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
41. Mosacism in two respects…
• In terms of WT vs. mutant alleles in an animal…
42. Mosacism in two respects…
• …and within the pool of modified genes
43. Screening strategy
• Sanger sequencing:
– Problematic
• Clone into plasmids:
Sanger sequence clones
• Deep sequencing:
another possibility
• Specific insertions:
– Internal-external primer
PCR
44. 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
45. 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)
46. 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
47. RNA quantification
• Nanodrop readings unreliable
• Qubit much better:
– Fluorescence-based
– More sensitive readings
– Available in shared equipment
51. 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)
52. • 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
53. 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
61. Injections: cytoplasmic vs. pronuclear
http://www.med.upenn.edu/cores/transgenic_and_chimeric_mouse_facility.shtml
62. 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
63. 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?
67. 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
68. 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
82. 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
87. Acknowledgements
VAI vivarium
Audra Guikema
Tina Meringa
Bryn Eagleson
Ashlee Verbis
Neil Brandow
Mat Rackham
Vivarium Staff
VAI
Dr. KwangHo Lee
Dr. Lorenzo Sempere
Dr. Bart Williams
Cassie Diegel
JAX
Michael Wiles
Wenning Qin
U of M GT Core
Dr. Thom Saunders
Elizabeth Hughes
The Mice
Notas do Editor
Mammalian codon optimized?
Mammalian codon optimized?
TALEN produced a point mutation; ZFN produced floxed allele
Get numbers to back up the relevance of this study!!!
a.) normal 20 vs. 30 and 30 w bp changes
b.) showing similar cleavage activities w the Surveyor assay
c.) showing processing of the 30 bp products into 20 bp products
a.) normal 20 vs. 30 and 30 w bp changes
b.) showing similar cleavage activities w the Surveyor assay
c.) showing processing of the 30 bp products into 20 bp products
a.) normal 20 vs. 30 and 30 w bp changes
b.) showing similar cleavage activities w the Surveyor assay
c.) showing processing of the 30 bp products into 20 bp products