Abstract
CRISPR/Cas9 system, an innovative method for genome manipulation, is being rapidly applied across organisms, including fishes, to understand basic physiology and gene function. CRISPR/Cas9 is a method for cutting double-stranded DNA across target sites. One of the critical steps in this method is confirming gene knockout of the organism, or cell, after genome modification, which causes insertions/deletions in the targeted gene of interest. The purpose of this study was to utilize CRISPR/Cas9 system (1) in vivo in Zebrafish (D. rerio) and (2) in vitro in Rainbow Trout (Oncorhynchus mykiss) hepatocytes and confirm knockouts through (1) Next-Generation Sequencing (NGS) and (2) real-time qPCR while exploring various in vitro transfection methods.
In vivo in Zebrafish:The Zebrafish genotypes were identified by next generation amplicon sequencing, using the MiSeq Illumina platform, to confirm mutation in the Zebrafish esr1 gene in Go (CRISPR mutated founder generation) individuals. Two regions were targeted in esr1, an antisense target (T1) in exon 1 and a sense target (T2) in exon 3 to increase the likelihood of gene disruption. Potential CRISPR founders (Go) were outcrossed with wild type individuals, embryos pooled, and DNA extracted. A total of 225 distinct mutant alleles were detected from the F1 progeny of the 45 G0 founders across the T1 and T2 regions that were sequenced. Thirty-eight fish had mutations at both target sites, two founders only possessed T1 mutations, and two other fish only possessed T2 mutations; three fish failed to show any mutation. Out of the 225 mutant alleles, the majority of indels that occurred were deletions. Of those, 160 deletions were detected (71.1% of mutant alleles) compared to 60 insertions (26.7% of mutant alleles) across the target regions. A total of five in-frame, substitutions occurred and were only detected in alleles across T2 (2.2% of mutant alleles). A total of 169 frameshifts (75.1% of mutant alleles) and 56 in-frame (24.9% of mutant alleles) mutations were detected. The results show that this mutation screening method is accurate in confirming specific CRISPR indels in the Zebrafish germline and produces high quality sequencing data without the need for cloning or additional sequencing steps.
In vitro in Rainbow Trout hepatocytes:Four CRISPR/Cas9 gRNAs were designed to target the A/B domains of the four Rainbow Trout estrogen receptors (ERs); ERα1, ERα2, ERβ1, and Erβ2. The A/B domain was selected because it contains dissimilar DNA sequence regions that are unique between the four ER’s and would prevent accidental targeting of more than one isoform at a time. Primary hepatocytes were isolated from juvenile Rainbow Trout. Lipofection and nucleofection were two methods utilized to transfect hepatocytes with CRISPR/Cas9 gRNA to target each ER isoform. Real-time qPCR was used to compare relative mRNA across control and experimental hepatocytes in triplicates. Since the CRISPR/Cas9 plasmid constructs contained a green fluorescent protein (GFP) reporter, hepatocytes were visualized using fluorescent microscopy to confirm transfection and transcription activation. Nucleofection of the 4.7 kb pmaxGFP plasmid control was successful as evidenced by positive GFP fluorescence. However, the larger 9.3 kb CRISPR/Cas9 plasmids for ERα1, ERα2, ERβ1, or ERβ2 did not successfully enter the hepatocytes (i.e., no positive GFP fluorescence). Similarly, another large 9.3 kb CRISPR/Cas9 plasmid for the vtg gene was also not successfully transfected. These results suggest a size limitation on plasmids transfected into Rainbow Trout hepatocytes using these methods, since the CRISPR/Cas9 plasmids were about 9.3 kb in size. In conclusion, more methods should be explored to develop a successful approach to difficult-to-transfect fish cells with a large CRISPR/Cas9 plasmid in this functional genomics approach.