4. See flyCRISPR (http://flycrispr.molbio.wisc.edu/) and
Addgene (http://www.addgene.org/crispr/oconnor-giles/)
for details about CRISPR designs. We used Addgene 51434
for generating CRIPSR donor plasmid for homology-directed
repair and Addgene 45946 for generating plasmids expressing
guide RNAs.
5. The stems and/or the linkers in the step loop series may
contain binding sites for tissue or temporal specific transcription factors; thus tagging the gene with stem-loop sequences
may influence its expression pattern. A quantitative comparison
between the expression pattern of the edited locus and the
endogenous gene is recommended. In the case where undesired ectopic expression occurs, stem or linker sequences
should be modified.
6. parS sequence from Burkholderia cenocepacia (J2315,
chr3:3440-3821, GB: AM747722) is used for genome labeling. The method is adapted from reference [12]. See Addgene
(https://www.addgene.org/Kerstin_Bystricky/) for more
information about Bystricky group plasmids.
7. To create heptane glue, cut ~100 cm of permanent Scotch
double-sided tape and put it into a 20 mL scintillation vial.
Add 15 mL of heptane. Rock the vial on a horizontal shaker
(100 rpm) for 24 h. Transfer the heptane into a 15 mL Corning
tube and centrifuge at 1000 Â g for 10 min. Take the supernatant and store at 4
C.
8. It is recommended to test the cutting efficiency of the two
target gRNA plasmids before homologous donor injection.
9. Homologous arms from the target genome should be
sequenced and used for constructing the integration donor
plasmid. Length of homologous arms can range from 0.5 to
1.5 kb.
10. Presence of more than 12 repeats of MS2 or PP7 stem-loops in
the 5
0 UTR of the tested genes (hb, Kr, kni, eve, and run)
caused gene inactivation as seen by the lack of phenotypic
rescue. In order to create viable tagged gene constructs able
to rescue a null mutant, it is recommended that the stem-loops
are integrated in the intron or in the 3
0 UTR.
11. A drying test before formal injections is recommended. An
empirical indication of appropriate drying time is that about
20% embryos leak when the needle shoves into the germplasm.
12. Balancer lines contain chromosomes with multiple inversions,
which prevent viable meiotic recombinations. This is particularly important for tracking and maintaining transgenes that
lack visible markers. See BDSC information about balancers
(https://bdsc.indiana.edu/stocks/balancers/index.html).
382
Hongtao Chen and Thomas Gregor
Addgene (http://www.addgene.org/crispr/oconnor-giles/)
for details about CRISPR designs. We used Addgene 51434
for generating CRIPSR donor plasmid for homology-directed
repair and Addgene 45946 for generating plasmids expressing
guide RNAs.
5. The stems and/or the linkers in the step loop series may
contain binding sites for tissue or temporal specific transcription factors; thus tagging the gene with stem-loop sequences
may influence its expression pattern. A quantitative comparison
between the expression pattern of the edited locus and the
endogenous gene is recommended. In the case where undesired ectopic expression occurs, stem or linker sequences
should be modified.
6. parS sequence from Burkholderia cenocepacia (J2315,
chr3:3440-3821, GB: AM747722) is used for genome labeling. The method is adapted from reference [12]. See Addgene
(https://www.addgene.org/Kerstin_Bystricky/) for more
information about Bystricky group plasmids.
7. To create heptane glue, cut ~100 cm of permanent Scotch
double-sided tape and put it into a 20 mL scintillation vial.
Add 15 mL of heptane. Rock the vial on a horizontal shaker
(100 rpm) for 24 h. Transfer the heptane into a 15 mL Corning
tube and centrifuge at 1000 Â g for 10 min. Take the supernatant and store at 4
C.
8. It is recommended to test the cutting efficiency of the two
target gRNA plasmids before homologous donor injection.
9. Homologous arms from the target genome should be
sequenced and used for constructing the integration donor
plasmid. Length of homologous arms can range from 0.5 to
1.5 kb.
10. Presence of more than 12 repeats of MS2 or PP7 stem-loops in
the 5
0 UTR of the tested genes (hb, Kr, kni, eve, and run)
caused gene inactivation as seen by the lack of phenotypic
rescue. In order to create viable tagged gene constructs able
to rescue a null mutant, it is recommended that the stem-loops
are integrated in the intron or in the 3
0 UTR.
11. A drying test before formal injections is recommended. An
empirical indication of appropriate drying time is that about
20% embryos leak when the needle shoves into the germplasm.
12. Balancer lines contain chromosomes with multiple inversions,
which prevent viable meiotic recombinations. This is particularly important for tracking and maintaining transgenes that
lack visible markers. See BDSC information about balancers
(https://bdsc.indiana.edu/stocks/balancers/index.html).
382
Hongtao Chen and Thomas Gregor