Synonymous codon massive reporter library in zebrafish embryos. 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. Overall design: 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.

信使RNA（Messenger RNA，mRNA）的稳定性对细胞内稳态基因表达水平具有显著影响。跨物种而言，mRNA稳定性受密码子组成的强烈影响，且呈现翻译依赖性，其相关机制被称为密码子最优性（codon optimality）。我们开发了iCodon（www.iCodon.org），这是一种可通过在编码序列中引入同义密码子替换来定制mRNA表达的算法。iCodon针对四种脊椎动物转录组进行了优化：小鼠、人类、蛙类与鱼类。用户可基于任意编码序列的密码子组成预测其mRNA稳定性，进而生成编码同一蛋白质的更稳定（优化型）或更不稳定（去优化型）变体。进一步而言，我们通过大规模报告基因文库证实，密码子最优性预测结果与mRNA稳定性显著相关；同时借助荧光报告基因及对斑马鱼胚胎和/或人类细胞内源基因表达的分析，证实该预测与基因表达水平存在关联。因此，iCodon将为基础生物学研究以及生物技术与生物医学领域的众多应用提供助力。

实验整体设计：我们设计了包含1600条序列的文库：其中包含100种不同的100氨基酸长度编码序列，这些序列覆盖了斑马鱼蛋白质组的平均组成；每种编码序列对应10种变体，均通过iCodon设计为5个稳定性预测梯度递增的组别，每个组别包含2条序列。这些组别被命名为iCodon 1至iCodon 5，对应mRNA预测稳定性从低到高依次提升。此外，我们通过IDT的密码子优化工具独立迭代5次，设计了5条同义序列；同时借助Genewiz的密码子优化工具设计了1条同义序列，因为该方法在每次独立运行中均会生成完全一致的序列。将上述序列克隆至11个固定密码子的下游，以控制翻译起始效率一致；所有报告基因均包含恒定的5'非翻译区（untranslated region，UTR）与3'非翻译区（untranslated region，UTR）序列。我们合成了1600条设计序列中的1395条，通过体外转录合成mRNA并注射至1细胞期斑马鱼胚胎中；随后通过靶向RNA测序（RNA-sequencing），计算注射后2小时、5小时与8小时的mRNA降解情况。