A New Strategy for Increasing Knock-in Efficiency: Multiple Elongase and Desaturase Transgenes Knock-in by Targeting Long Repeated Sequences.

CRISPR/Cas9-mediated knock-in (KI) has a wide application in gene therapy, gene function study, and transgenic breeding programs. Unlike gene therapy, which requires accurate KI to correct gene mutation, transgenic breeding programs can accept robust KI as long as integration does not interrupt normal gene functions and result in any negative pleiotropic effects. High KI efficiency is required to reduce the breeding cost and shorten the breeding period, especially in transferring multiple foreign genes to a single individual. To elevate the KI efficacy and achieve multiple gene KIs simultaneously, we introduced a new strategy that enables transgene integration into numerous sites of the genome by targeting long repeated sequences (LRSs). Using this simple strategy, for the first time we successfully generated transgenic fish carrying the masu salmon () gene and rabbitfish () Δ4 and Δ6 genes, and achieved robust target KI of and Δ6 genes at multiple sites of LRS1 and LRS3, respectively, in the initial generation. This demonstrated that donor plasmid homology arms, which were nearly identical but not completely the same as the genome sequence, still led to on-target KI. Although the target KI efficiencies at LRS1, LRS2, and LRS3 sites were still relatively low in the current study, it is very promising that 100% KI efficiency in the future could be realized and perfected by selection of better LRSs and optimization of sgRNAs.