While loss-of-function approaches have included gene knock-down using morpholino-modified oligonucleotides and random mutagenesis using mutagens or retroviruses, more recent approaches include targeted mutagenesis using zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology.  To facilitate evaluation of toxic mechanisms for important categories of aryl hydrocarbon pollutants, whose actions are known to be receptor mediated, we used ZFN and CRISPR-Cas9 approaches to generate aryl hydrocarbon receptor 2a (AHR2a) and AHR2b gene mutations in Atlantic killifish (Fundulus heteroclitus) embryos.  We first used five-finger ZFNs targeting exons 1 and 3 of AHR2a.  We successfully induced frameshift mutations in AHR2a exon 3 with ZFN and CRISPR-Cas9 guide RNAs, with mutation frequencies of 10% and 16%, respectively. 
ZFN
While loss-of-function approaches have included gene knock-down using morpholino-modified oligonucleotides and random mutagenesis using mutagens or retroviruses, more recent approaches include targeted mutagenesis using zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. 
TALENs
To facilitate evaluation of toxic mechanisms for important categories of aryl hydrocarbon pollutants, whose actions are known to be receptor mediated, we used ZFN and CRISPR-Cas9 approaches to generate aryl hydrocarbon receptor 2a (AHR2a) and AHR2b gene mutations in Atlantic killifish (Fundulus heteroclitus) embryos.  Subsequently, CRISPR-Cas9 guide RNAs were designed to target regions in exon 2 and 3 of AHR2a and AHR2b.  We successfully induced frameshift mutations in AHR2a exon 3 with ZFN and CRISPR-Cas9 guide RNAs, with mutation frequencies of 10% and 16%, respectively.  In AHR2b, mutations were induced using CRISPR-Cas9 guide RNAs targeting sites in both exon 2 (17%) and exon 3 (63%).  We screened AHR2b exon 2 CRISPR-Cas9-injected embryos for off-target effects in AHR paralogs.  No mutations were observed in closely related AHR genes (AHR1a, AHR1b, AHR2a, AHRR) in the CRISPR-Cas9-injected embryos. 
CRISPR-Cas9