1. WORKSHOP: CRISPR in insects
(Christine Merlin, Texas A&M University)

2. Considerations for single guide RNA design
Egg microinjections
Mutation detection strategies

3. Generate a DSB at a specific-site within the genome
Targeted genome editing
ZFNs/TALENs/CRISPR
Generate a DSB at a specific-site within the genome
Induced DSB
Desired or random site-specific modification
Cell’s endogenous DNA repair machinery
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

4. 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)

5. 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

6. 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)

7. Designing your single guide RNAs
Softwares available online

8. Designing your single guide RNAs
Ex.

9. Germline targeting by egg microinjection at a
which embryonic development stage?
Bombyx mori
Anterior
Yolk
Germline
precursors
Posterior
Syncitial preblastoderm

10. Germline targeting by egg microinjection at a
which embryonic development stage?
Bombyx mori
Monarch butterfly
Anterior
Anterior
Micropyle: Location of
sperm entry
Yolk
Yolk
Pronucleus male
Pronucleus female
Zygote nucleus
Germline
precursors
Posterior
Posterior
Syncitial preblastoderm
“One nucleus” stage

11. Which strategies to detect mutants?
Many options are available but the choice may depend on the
model organism
Knock-out
Knock-in
 Restriction site
 Simple PCR
 T7 Endonuclease I assay
 Cas9-based cleavage assay
 High Resolution Melt Analysis
 Fluorescent reporter tag
 Restriction site

12. Summary of targeting and screening strategies
Egg injection strategy
Selection strategy
ZFN mRNAs
Cas9 mRNAs
+ guide RNA
AAAAAAAA
TALEN mRNAs
AAAAAAAA
Micropyle
Selection of highly
targeted mosaics
for backcross
Co-inject one nucleus stage embryos (20min AEL)
Fertilized egg
gDNA
Assay of somatic mosaicism in individual larvae
Assay lesions
at targeted site in larva

13. Detection of CRISPR-mediated mutagenic lesions
using gRNAs with restriction enzyme cut site
Uncut
Lesions
Restriction enzyme
cut site
…but you may want to use the most efficient sgRNA
regardless of the presence of an RE cut site

14. Detection of CRISPR-mediated mutagenic lesions
using a T7E1 in vitro cleavage assay
T7 Endonuclease I recognizes and cleaves non-perfectly matched DNA 1. 2. 3. 4.
Requirements:
No SNP present in the amplicon
- Work with isogenic lines
- Knowledge of SNP at locus
of interest in the population
Potential problems:
- Not quantitative
- T7E1 enzyme is not absolutely mismatch specific; it also nicks
dsDNA slowly.
Cleaves heteroduplexes

15. 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).

16. Detection of CRISPR-mediated mutagenic lesions
using a Cas9-based in vitro cleavage assay
See Markert et al, 2016 for details on the assay

17. 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.

18. 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): e doi: /journal.pone

19. 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

20. Detection of CRISPR-mediated mutagenic lesions
using a simple PCR-based protocol
Yu et al, 2014, PLoS ONE
9(6): e98282.

21. Detection of CRISPR-mediated mutagenic lesions
using a simple PCR-based protocol
Once line
established
Yu et al, 2014, PLoS ONE
9(6): e98282.

22. 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

23. Detection of CRISPR-mediated mutagenic lesions
using the restriction site present in the KI cassette KI
Uncut
No KI
Lesions KI
control
Mosaics
KI events

24. 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

25. 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

26. Vectors to be found on Addgene

27. 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
bp for TALENs
 Targeted site can be chosen to span
an endogenous restriction (in the spacer)

28. cleaves heteroduplexes
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/re-annealing 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)
85C to 25C (-0.1C/sec)
annealing
- Add 1ul T7 Endonuclease I (15min at 37C)
- Bead purify and run on gel
T7 Endonuclease I
cleaves heteroduplexes