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While global knockouts are extremely straightforward to make, a potentiallymore useful approach is to generate knockouts in specific tissues. This strategy can
be particularly useful for genes that are globally expressed or have unique functions
in different tissues. There are two strategies for generating tissue specific Cas9-
mediated genomic changes. Both rely on generating transgenic fish, which stably
express Cas9. In one approach, a single Tol2 transposon expresses both Cas9 and
the targeting sgRNA [ 47 ]. This single DNA construct makes it possible to inactivate
a target gene in a specific tissue in one generation, which is additionally facilitated
by the inclusion of a fluorescent marker for screening of carriers of the transgene.
Cas9 is expressed under a tissue-specific promoter and the sgRNA is expressed
ubiquitously using a U6 splicesomal RNA promoter. The authors of this system
have made generating the transposon even more straightforward by modifying it so
that it can be used with the Gateway cloning system [ 48 ]. Thus combining different
promoters and sgRNAs becomes simply a matter of mixing appropriate plasmids
together for a mix and match approach.
The second approach requires generating two different Tol2 transposons and twotransgenic lines, which are then mated with one another [ 49 ]. One transposon
expresses a tissue-specific or otherwise regulatable Cas9 gene and the other one
expresses one or more sgRNA molecules under the control of distinct U6 promot-
ers. The identification of transgenic carriers is again facilitated by the presence of
fluorescent markers in both transposons. Once carriers are identified, crossbreeding
of the different transgenic lines introduces mutations in specific Cas9-expressing
tissues. This system is slower than using a single transposon, since two different
transgenic lines need to be identified, but it is advantageous to express multiple
sgRNAs at once to ensure that a loss of function allele is identified in the target gene
of interest. These tissue-specific transgenic systems are still relatively new, but they
hold promise for being used in many different studies. Continued improvements to
these systems will come from additional conditional Cas9 activation strategies,
such as small molecules or light, which are rapidly being developed and optimized
[ 50 , 51 ].
Several recent studies have used the new genome editing strategies in zebraf-ish to study retina and RPE [ 52 – 58 ]. Many of these studies are already exploiting
the speed and ease by which CRISPR/Cas9 can be used to generate mutant
alleles. Zebrafish are well known for distinct experimental advantages. Genetic
screens have been used to identify many genes essential for photoreceptor func-
tion [ 59 , 60 ] and development [ 61 , 62 ]. Recent work has further exploited the
transparency of larvae and the ease of generating transgenic strains to conduct
sophisticated imaging experiments using fluorescent markers. These studies,
which are too numerous to list all here, have provided insight into many aspects
of photoreceptor biology. They include analyses of photoreceptor behavior dur-
ing development [ 63 – 65 ], regeneration [ 66 , 67 ] and disease [ 68 , 69 ]. They also
provide insight into intracellular events in photoreceptors such as autophagy [ 21 ,
70 ], protein trafficking [ 71 , 72 ] and dynamics of essential second messenger mol-
ecules such as Ca2+ [ 24 , 73 ]. The recent rapid development of sophisticated
genome editing techniques ensures that zebrafish will continue to provide novel
S.E. Brockerhoff