Download T7 Endonuclease I assay

WORKSHOP: CRISPR in insects
(Christine Merlin, Texas A&M University)
Considerations for single guide RNA design
Egg microinjections
Mutation detection strategies
Targeted genome editing
ZFNs/TALENs/CRISPR
Generate a DSB at a specific-site
within the genome
Induced DSB
Cell’sorendogenous
DNA repair machinery
Desired
random site-specific
modification
KNOCK-IN
KNOCKOUT
Homologous
recombination
Non-homologous
end joining
Sister chromatid
Donor
DNA (plasmid)
Perfect repair
Imperfect repair
Deletions
Insertions
Frame-shifts and truncated proteins
The CRISPR/Cas9 revolution:
Programmable RNA-guided DNA endonucleases
 Emerged as a potentially facile and efficient alternative to ZFNs and TALENs
for gene targeting in 2013.
 In only a few years, more than 1100 papers have been published using CRISPR
for gene targeting (55 in insects).
 CRISPR-mediated gene targeting relies on:
- A target-specific gRNA sequence tethered
to a tracrRNA
- A Cas9 protein or Cas9 encoding mRNA
- Target sequence complementary to the
gRNA, which should be followed by a
Protospacer Adjacent Motif (PAM)
Designing your single guide RNAs
 Choosing an appropriate target sequence in the genomic DNA
 20 nucleotides followed by the appropriate Protospacer Adjacent Motif (PAM):
- For Cas9 to successfully bind to DNA, the target sequence in the genomic
DNA must be complementary to the gRNA sequence and must be immediately
followed by the correct protospacer adjacent motif or PAM sequence.
- The PAM sequence is present in the DNA target sequence but not in the gRNA
sequence.
The Protospacer Adjacent Motif (PAM) Sequence is essential for cleavage
Designing your single guide RNAs
TIP: Pay attention to the origin of the Cas9 used, as the PAM sequence
differs as a function of the species
When using SpCas9, potential target sites are both (5’-20nt-NGG) and (5’-CCN-20nt)
Designing your single guide RNAs
Softwares available online
Designing your single guide RNAs
Ex.
Germline targeting by egg microinjection at a
which embryonic development stage?
Bombyx mori
Anterior
Yolk
Germline
precursors
Posterior
Syncitial preblastoderm
Germline targeting by egg microinjection at a
which embryonic development stage?
Bombyx mori
Monarch butterfly
Anterior
Anterior
Micropyle: Location of
sperm entry
Pronucleus male
Pronucleus female
Zygote nucleus
Yolk
Yolk
Germline
precursors
Posterior
Syncitial preblastoderm
Posterior
“One nucleus” stage
Which strategies to detect mutants?
Many options are available but the choice may depend on the
model organism
Knock-out
 Restriction site
 Simple PCR
 T7 Endonuclease I assay
 Cas9-based cleavage assay
 High Resolution Melt Analysis
Knock-in
 Fluorescent reporter tag
 Restriction site
Summary of targeting and screening strategies
Egg injection strategy
ZFN mRNAs
AAAAAAAA
AAAAAAAA
Selection strategy
Cas9 mRNAs
+ guide RNA
TALEN mRNAs
AAAAAAAA
AAAAAAAA
Micropyle
Selection of highly
targeted mosaics
for backcross
Co-inject one nucleus stage
embryos (20min AEL)
gDNA
Fertilized egg
Assay of somatic mosaicism in
individual larvae
Assay lesions
at targeted site in larva
Detection of CRISPR-mediated mutagenic lesions
using gRNAs with restriction enzyme cut site
Uncut
Uncut
EagI
cry2 exon 2
Lesions
Cut with ZFN
cry2 exon 2
Restriction enzyme
cut site
Control
Injected w/ both
ZFN mRNAs
No
EagI EagI
…but you may want to use the most efficient sgRNA
regardless of the presence of an RE cut site
No
EagI EagI
Detection of CRISPR-mediated mutagenic lesions
using a T7E1 in vitro cleavage assay
T7 Endonuclease I recognizes and cleaves non-perfectly matched DNA
1.
Requirements:
No SNP present in the amplicon
- Work with isogenic lines
- Knowledge of SNP at locus
of interest in the population
2.
3.
Cleaves heteroduplexes
4.
Potential problems:
- Not quantitative
- T7E1 enzyme is not absolutely
mismatch specific; it also nicks
dsDNA slowly.
Detection of CRISPR-mediated mutagenic lesions
using a T7E1 in vitro cleavage assay
What I personally do not like: qualitative but not quantitative
(does not allow to select mosaics with high levels of targeting).
Detection of CRISPR-mediated mutagenic lesions
using a Cas9-based in vitro cleavage assay
See Markert et al, 2016 for details on the assay
Detection of CRISPR-mediated mutagenic lesions
using High Resolution Melt Analysis (qPCR)
HRMA measures differences in the melt curves between amplified fragments.
No SNP allowed
The differences may be small
depending on the sequence change.
Specialized software may be required
to detect mutated samples.
Less labor intensive than Cas9-based
assay but may be less appropriate for
non-model species as SNPs should be
avoided.
Figure 1. 100 bp amplicons are optimal for HRM detection of nuclease induced small indels.
Thomas HR, Percival SM, Yoder BK, Parant JM (2014) High-Throughput Genome Editing and Phenotyping Facilitated by High Resolution Melting
Curve Analysis. PLoS ONE 9(12): e114632. doi:10.1371/journal.pone.0114632
http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0114632
Detection of CRISPR-mediated mutagenic lesions
generated using multiple sgRNAs
2 or more sgRNAs
 2 or more sgRNAs can be used
 Genomic deletions can be screened by PCR
 Potential problem: Increase of off target effects
Potential problem: Increase of off target effects
Chen et al, Scientific Reports, 2014
Detection of CRISPR-mediated mutagenic lesions
using a simple PCR-based protocol
Yu et al, 2014, PLoS ONE
9(6): e98282.
Detection of CRISPR-mediated mutagenic lesions
using a simple PCR-based protocol
Once line
established
Yu et al, 2014, PLoS ONE
9(6): e98282.
Detection of CRISPR-mediated mutagenic lesions by
knock-in of a stop codon cassette containing
a restriction site
Advantage: Provide control over
ORF truncation.
Screen by:
PCR using
- 1 primer specific to the cassette
- 1 primer on genomic DNA
OR
Using a restriction assay
Gagnon et al, PLOS One, 2014
Detection of CRISPR-mediated mutagenic lesions
using the restriction site present in the KI cassette
Uncut
KI
Uncut
EagI
cry2 exon 2
NoLesions
KI
Cut with ZFN
cry2 exon 2
KI
control
Control
Injected w/ both
Mosaics
ZFN
mRNAs
No
EagI EagI
No
EagI EagI
KI events
Beyond knockouts and knock-ins
Use of the Cas9 endonuclease as a DNA-binding domain
to activate or repress genes
- Activation by tethering the VP64 transcriptional activator
Beyond knockouts and knock-ins
Use of the Cas9 endonuclease as a DNA-binding domain
to activate or repress genes
- Repression by occupancy of RNA PolII site using a catalytically inactive Cas9
Vectors to be found on Addgene
ZFNs and TALENs : Chimeric restriction
endonucleases engineered to cleave at a specific-site
 Function as a pair
 Each monomer is composed of:
- DNA-binding domain
- Cleavage domain (FokI Nuclease)
 Spacers requirement different:
- 5-7 bp for ZFNs
- 12-21 bp for TALENs
 Targeted site can be chosen to span
an endogenous restriction (in the spacer)
Assays to screen mutations
Genotyping for the presence of lesions using a T7 Endonuclease I assay.
Principle:
 T7 Endonuclease I recognizes
and cleaves non-perfectly matched
DNA.
 PCR your target (ex. mix of WT
and mutated products) and
proceed to denaturation/reannealing before subjecting your
products to T7 endonuclease I.
- 200ng DNA with 2ul 10X NEB2 Buffer (vf=19ul)
95C 5minutes
denaturation
95C to 85C (-2C/sec)
annealing
85C to 25C (-0.1C/sec)
T7 Endonuclease I
cleaves heteroduplexes
- Add 1ul T7 Endonuclease I (15min at 37C)
- Bead purify and run on gel