target sites can be used to delete up to 20 kb genomic region if
genomic modifications, such as deletions and point mutations,
are desired in the following experiments. Depending on the
locus of interest, the deleted region may include coding
sequences and/or cis-regulatory elements.
2. Sequence the gRNA recognition sites from the target genome
to avoid point mutations.
3. Generate plasmids that express the gRNAs [22] (see Note 8).
4. Clone the flanking homologous regions into the donor plasmid, in which a phenotypic selection marker flanked with two
attP sites is located between the two homologous arms (see
Note 9). The attP sites are designed for introducing stemloops or parS tags through RMCE.
5. Set up embryo collection cages with germline-supplied Cas9
females and males carrying the target genome. Embryos collected from these cages are used for fly transformation.
6. Follow Subheading 3.2 to generate transgenic flies with the
targeted deletion and integrated attP sites by co-injecting the
gRNA-expressing plasmid (or two plasmids if two gRNA target
sites are selected) and the homologous donor plasmid to
embryo germline.
cas9; +/+
cas9; +/+
+/Y; RFP + /+
yw; CyO/Sp
cas9/Y; +/+
yw; CyO/Sp
C31; +/+
yw; CyO/RFP +
C31/Y; RFP + /+
yw; CyO/Sp
yw; MS2(RFP - )/CyO or Sp
HA1
HA2
HA1
HA2
RFP
P
P
RFP
P
P
B
B
PAM
PAM
HA1
HA2
exon
exon
MS2
exon
exon
CRISPR donor plasmid
RMCE plasmid
Injection (CRISPR donor
+ gRNA plasmids)
Injection
(RMCE plasmid)
Screen for RFP
in the eyes
Stock expansion
Screen for loss of RFP
in the eyes
exon MS2
exon
a
b
Step 1
Step 2
Step 1
Step 2
Fig. 1 Genomic integration of live imaging tags through CRISPR-Cas9-based genome editing and attB/attP
cassette exchange. (a) A two-step approach to insert MS2 stem-loops in the intron of a target gene. In the first
step, the intron and the adjacent exons are replaced by an eye-expressing RFP marker flanked by two attP
sites (P) through CRISPR-mediated homologous recombination. PAM, protospacer adjacent motif. HA, homologous arm. In the second step, the deleted genomic sequence with an MS2 tag in the intron is inserted
between two attB (B) sites and introduced back to the genome through attB/attP recombinations. The same
method also applies to other imaging tags (PP7 or parS). (b) Crossing scheme to generate transgenic flies
through the two-step integration approach. A target gene on the second chromosome is shown as an example.
Appropriate balancers should be used for target genes on the other chromosomes (see Note 12)
376
Hongtao Chen and Thomas Gregor
genomic modifications, such as deletions and point mutations,
are desired in the following experiments. Depending on the
locus of interest, the deleted region may include coding
sequences and/or cis-regulatory elements.
2. Sequence the gRNA recognition sites from the target genome
to avoid point mutations.
3. Generate plasmids that express the gRNAs [22] (see Note 8).
4. Clone the flanking homologous regions into the donor plasmid, in which a phenotypic selection marker flanked with two
attP sites is located between the two homologous arms (see
Note 9). The attP sites are designed for introducing stemloops or parS tags through RMCE.
5. Set up embryo collection cages with germline-supplied Cas9
females and males carrying the target genome. Embryos collected from these cages are used for fly transformation.
6. Follow Subheading 3.2 to generate transgenic flies with the
targeted deletion and integrated attP sites by co-injecting the
gRNA-expressing plasmid (or two plasmids if two gRNA target
sites are selected) and the homologous donor plasmid to
embryo germline.
cas9; +/+
cas9; +/+
+/Y; RFP + /+
yw; CyO/Sp
cas9/Y; +/+
yw; CyO/Sp
C31; +/+
yw; CyO/RFP +
C31/Y; RFP + /+
yw; CyO/Sp
yw; MS2(RFP - )/CyO or Sp
HA1
HA2
HA1
HA2
RFP
P
P
RFP
P
P
B
B
PAM
PAM
HA1
HA2
exon
exon
MS2
exon
exon
CRISPR donor plasmid
RMCE plasmid
Injection (CRISPR donor
+ gRNA plasmids)
Injection
(RMCE plasmid)
Screen for RFP
in the eyes
Stock expansion
Screen for loss of RFP
in the eyes
exon MS2
exon
a
b
Step 1
Step 2
Step 1
Step 2
Fig. 1 Genomic integration of live imaging tags through CRISPR-Cas9-based genome editing and attB/attP
cassette exchange. (a) A two-step approach to insert MS2 stem-loops in the intron of a target gene. In the first
step, the intron and the adjacent exons are replaced by an eye-expressing RFP marker flanked by two attP
sites (P) through CRISPR-mediated homologous recombination. PAM, protospacer adjacent motif. HA, homologous arm. In the second step, the deleted genomic sequence with an MS2 tag in the intron is inserted
between two attB (B) sites and introduced back to the genome through attB/attP recombinations. The same
method also applies to other imaging tags (PP7 or parS). (b) Crossing scheme to generate transgenic flies
through the two-step integration approach. A target gene on the second chromosome is shown as an example.
Appropriate balancers should be used for target genes on the other chromosomes (see Note 12)
376
Hongtao Chen and Thomas Gregor