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 (Oncorhynchus masou) elovl2 gene and rabbitfish (Siganus
canaliculatus) Δ4 fad and Δ6 fad genes, and achieved robust target KI of elovl2 and Δ6 fad 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.