An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters

CRISPR/Cas9 gene editing is effective in manipulating genetic loci in mammalian cell cultures and whole fish but efficient platforms applicable to fish cell lines are currently limited. Our initial attempts to employ this technology in fish cell lines using heterologous promoters or a ribonucleoprotein approach failed to indicate genomic alteration at targeted sites in a tilapia brain cell line (OmB). For potential use in a DNA vector approach, endogenous tilapia beta Actin (OmBAct), EF1 alpha (OmEF1a), and U6 (TU6) promoters were isolated. The strongest candidate promoter determined by EGFP reporter assay, OmEF1a, was used to drive constitutive Cas9 expression in a modified OmB cell line (Cas9-OmB1). Cas9-OmB1 cell transfection with vectors expressing gRNAs driven by the TU6 promoter achieved mutational efficiencies as high as 81% following hygromycin selection. Mutations were not detected using human and zebrafish U6 promoters demonstrating the phylogenetic proximity of U6 promoters as critical when used for gRNA expression. Sequence alteration to TU6 improved mutation rate and cloning efficiency. In conclusion, we report new tools for ectopic expression and a highly efficient, economical system for manipulation of genomic loci and evaluation of their causal relationship with adaptive cellular phenotypes by CRISPR/Cas9 gene editing in fish cells.


Construction of EGFP reporter vectors
A modified pBluescript II SK(+) plasmid was generated to have an NcoI site in the multiple cloning site. The EGFP coding sequence along with the SV40 poly(A) terminator sequence was PCR amplified with EGFP_F1 and EGFP_R1(with XmaI and SacI site 5' extension) primers from pENTR5'_ubi:loxP-EGFP-loxP (Addgene #27322) plasmid template and cloned into the modified pBluescript vector with NcoI and SacI enzymes to generate the following base EGFP vector (EGFP_SV40PA): Supplementary Figure S2.
The OmEF1a amplicon was re-PCR amplified with EF1a_F3 (to add a SalI site to the 5' end) and EF1_R3b (single nucleotide change in this primer generated a NcoI site at the start codon) primers. The OmBAct and OmEF1a promoters were cloned into EGFP_SV40PA with SalI and NcoI enzymes. The CAG promoter was digested from the pSpCas9(BB)-2A-Puro (Addgene # 48139) plasmid. The CMV promoter was PCR amplified from pCS2-nCas9n (Addgene # 47929) plasmid with CMV_F2 and CMV_R1 primers. To remove an internal NcoI site from the SV40 promoter, site directed mutagenesis was used to change a G to a C (-171 from the start codon). The SV40 promoter was first PCR amplified in two fragments, the 5' end (with SV40_F1 and SV40_SDM_R1 primers) and the 3' end (with SV40_SDM_F1 and SV40_R6b2 primers) from the pBABE-hygro-hTERT (Addgene # 1773) plasmid. The two fragments were assembled into the full length SV40 with a stitch PCR reaction using SV40_F1 and SV40_R6b2 primers. The full length CAG, CMV, and SV40 fragments were cloned into EGFP_SV40PA with KnpI and NcoI enzymes. The Zubi promoter was digested from the pENTR5'_ubi:loxP-EGFP-loxP (Addgene #27322) plasmid with XhoI and NcoI and ligated into EGFP_SV40PA digested with SalI and NcoI enzymes (XhoI and SalI have complimentary cohesive ends). Example of resulting constructs (OmEFaEGFP_SV40PA) is shown in Supplementary Fig. S3.

Construction of EF1aCas9P2APuro vector
A new P2A sequence was synthesized by annealing two oligos (P2A_F1 and P2A_R1) with a 14 bp complimentary region between them followed by filling in of the single stranded regions with DNA polymerase and dNTPs to generate the following P2A DNA fragment with new restriction sites (SpeI 5' end, NcoI and SacI 3' end) on the ends: This fragment was cloned into pBluescript II SK(+) plasmid using SpeI and SacI enzymes to make the pBSP2A plasmid. The puromycin resitance gene (with the bGH poly(A) signal) was PCR amplified from pSBbi-GP (Addgene # 60511) plasmid, digested with BspHI and SacI and ligated into pBSP2A digested with NcoI and SacI enzymes (BspHI and NcoI have complementary cohesive ends). The P2APuro fragment was digested from the pBSP2APuro plasmid and ligated into the pSBbi-GP (Addgene # 60511) plasmid backbone (including the Sleeping Beauty ITRs) with XhoI and NdeI enzymes making the pSBP2APuro plasmid. The pSBP2APuro plasmid was digested with PvuII and XhoI, blunted with New England Biolabs Quick Blunting kit (cat# E1201S), and re-ligated to remove excess unnecessary DNA sequence. The Cas9 coding sequence was digested from the pCS2-nCas9n (Addgene # 47929) plasmid with SalI and AgeI enzymes and ligated into the pSBP2APuro plasmid digested with SalI and XmaI enzymes (AgeI and XmaI have complementary cohesive ends) to make the Cas9P2APuroSB plasmid. The OmEF1a promoter was cloned into the Cas9P2APuroSB plasmid using SalI and NcoI enzymes to complete the OmEF1aCas9P2APuroSB construct.

Construction of the TU6 template vector
The original TU6 promoter PCR amplicon was re-PCR amplified with TU6_1b and TU6_R1b primers (to add restriction sites) and cloned into pBluescript II SK(+) base vector XhoI and EagI enzymes. To make a single nucleotide change at the -273 (A to C) and remove an internal AgeI restriction site, the 5' 140 bp of TU6 was removed from this vector by AgeI and XhoI digestion and replaced with a PCR amplicon of the same 140 bp (amplified with TU6_1b and TU6_R3) digested with XhoI and XmaI (ligation of AgeI and XmaI complimentary cohesive ends destroyed the original AgeI recognition site). This vector (pBSTU6) was then used as template DNA for subsequent PCR reactions to generate expression cassettes of individual gRNAs upon cloning into the gRNAscaffHygroR vector.

Construction of the gRNAscaffHygroR vector
The Hygromycin resistance gene was PCR amplified from pBABE-hygro-hTERT (Addgene # 1773) plasmid using Hygro_F1 and Hygro_R1 primers, digested with BspHI and NotI and cloned into the OmEF1aEGFP_SV40PA vector (see above) digested with NcoI and NotI (BspHI and NcoI have complimentary cohesive ends), replacing the EGFP coding sequence. The modified guide RNA scaffold sequence was PCR amplified from gRNA_GFP-T2 (Addgene # 41820 ) plasmid using gRNAscaff_F2 and gRNAscaff(SacI)_R primers and cloned into the vector with XmaI and SacI enzymes.