Synonymous codon massive reporter library in zebrafish embryos

Messenger RNA (mRNA) stability substantially impacts steady-state gene expression levels in a cell. mRNA stability is strongly affected by codon composition in a translation-dependent manner across species, through a mechanism termed codon optimality. We have developed iCodon (www.iCodon.org), an algorithm for customizing mRNA expression through the introduction of synonymous codon substitutions into the coding sequence. iCodon is optimized for four vertebrate transcriptomes: mouse, human, frog, and fish. Users can predict the mRNA stability of any coding sequence based on its codon composition and subsequently generate more stable (optimized) or unstable (deoptimized) variants encoding for the same protein. Further, we show that codon optimality predictions correlate with both mRNA stability using a massive reporter library and expression levels using fluorescent reporters and analysis of endogenous gene expression in zebrafish embryos and/or human cells. Therefore, iCodon will benefit basic biological research, as well as a wide range of applications for biotechnology and biomedicine. We designed a library of 1,600 sequences containing 100 different 100 amino acid long coding sequences capturing the average composition of the zebrafish proteome, with 10 variants each designed using iCodon to have 5 bins of increasing predicted stability with 2 sequences in each bin. The groups were referred as iCodon 1, iCodon 2, iCodon 3, iCodon 4, and iCodon 5, from less to more stable mRNA predictions. Additionally, 5 synonymous sequences were designed using 5 independent iterations of IDT’s codon optimization tool and 1 synonymous sequence using Genewiz’s codon optimization tool as this method returned the exact same sequences in each independent run. These sequences were cloned downstream of 11 fixed codons to control for similar translation initiation and the reporters contain constant 5’ and 3’ UTR sequences. 1395 of the 1600 designed sequences were ordered, in vitro synthetized mRNA was injected into 1-cell stage zebrafish embryos, and the mRNA decay after 2, 5, and 8 hours post injection was calculated by targeted RNA-sequencing.