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2015 ; 12
(6
): 535-40
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Rapid reverse genetic screening using CRISPR in zebrafish
#MMPMID25867848
Shah AN
; Davey CF
; Whitebirch AC
; Miller AC
; Moens CB
Nat Methods
2015[Jun]; 12
(6
): 535-40
PMID25867848
show ga
Identifying genes involved in biological processes is critical for understanding
the molecular building blocks of life. We used engineered CRISPR (clustered
regularly interspaced short palindromic repeats) to efficiently mutate specific
loci in zebrafish (Danio rerio) and screen for genes involved in vertebrate
biological processes. We found that increasing CRISPR efficiency by injecting
optimized amounts of Cas9-encoding mRNA and multiplexing single guide RNAs
(sgRNAs) allowed for phenocopy of known mutants across many phenotypes in
embryos. We performed a proof-of-concept screen in which we used intersecting,
multiplexed pool injections to examine 48 loci and identified two new genes
involved in electrical-synapse formation. By deep sequencing target loci, we
found that 90% of the genes were effectively screened. We conclude that CRISPR
can be used as a powerful reverse genetic screening strategy in vivo in a
vertebrate system.
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