密碼子使用偏移是指各同義密碼子之間有著不同的使用率。細胞中共有64種密碼子，個別應對20種氨基酸，一種氨基酸可由不同的密碼子所轉譯而成，而這些密碼子彼此稱為同義密碼子。細胞在進行轉譯的過程中，對於同義密碼子的選擇並不是隨機的，而是有所偏好選擇地去使用這些同義密碼子，這個現象我們稱為密碼子使用偏移。近期知道密碼子使用偏移會影響細胞進行轉譯時延伸的速度，當最佳密碼子（常用密碼子）進行轉譯時，相應的tRNA進入核醣體A位點將更快，導致蛋白質合成速率的提升。相反地，當使用非最佳密碼子（不經常使用的密碼子）進行轉譯時，預期上核醣體解碼速率會相對較低。有趣的是最近的幾項研究表明，轉錄起始以及mRNA的穩定性與CUB高度相關。為了更好地了解CUB對mRNA表達水平的影響，我們決定使用具有強大基因敲除工具的秀麗隱桿線蟲（秀麗隱桿線蟲）作為動物模式系統來了解CUB對基因表達的影響。我們計算了秀麗隱桿線蟲中所有蛋白質編碼基因的密碼子偏移指數（CBI），並藉由選擇的具有高CBI和易於使用肉眼觀察之表現型的基因去篩選目標基因。根據上述這些規則，我們發現him-5是一個良好的編輯目標。根據秀麗隱桿線蟲密碼子使用表，將him-5的開放閱讀框架（ORF）做同義密碼子替換以降低CBI值，即密碼子去優化him-5（him-5-deop）。我們使用CRISPR / Cas9技術將gfp序列敲入原始的him-5基因座，以避免後續構建體的不完全基因替換，隨後再用his-5-deop替換gfp序列以監測CUB在mRNA表達中的影響。從基因分型和表型觀察的結果顯示，我們選擇同型合子gfp敲入品系進行第二次敲入。儘管基因分型結果表明gfp被敲入了him-5基因座，但定序結果顯示，Him-5基因座上的3'gRNA位點被刪除之外，且保留了him-5外顯子1。該結果表明CRISPR / Cas9系統可能無法使用相同的位點進行連續的CRISPR基因組編輯，因此每次敲入後應該突變PAM結構域以避免Cas9重複切割。我們仍在努力獲得新的基因編輯線蟲品系，儘管該模式動物尚未建立完成，但它將成為研究CUB在高等真核生物中影響mRNA表達水平中的新的重要的研究工具。

Codon usage bias (CUB) refers to the different usage frequency between synonymous codons. In specific, during protein translation, the selection of synonymous codons is not random, meaning certain synonymous codons are used with preference, and this phenomenon is called CUB. Past research showed that CUB affects the speed of translation elongation in both prokaryotes and eukaryotes. When the optimal codons (frequently used codons) are translated, the entering of the corresponding tRNAs into the ribosome A site will be faster, leading to faster protein synthesis rate. Conversely, when the non-optimal codons (infrequently used codons) are translated, slower ribosome decoding rate is expected. Intriguingly, several recent studies showed that the initiation of transcription as well as the stability of the mRNAs are highly correlated with CUB. In order to better understand the influences of CUB on mRNA expression levels, we decided to use Caenorhabditis elegans (C. elegans), which has a powerful gene knock-down tool, as the model system to understand the impact of CUB on gene expression. We calculated the codon bias index (CBI) of all protein coding genes in C. elegans and selected genes that have both high CBI and tractable phenotypes. With this rule, we found him-5 as a target. According to C. elegans codon usage table, him-5 open reading frame (ORF) was synonymously modified to lower the CBI values, namely codon de-optimized him-5 (him-5-deop). Then, I knocked gfp into the original him-5 locus using CRISPR/Cas9 technology to avoid incomplete gene replacement for the later constructs. Subsequently, the sequence of gfp was replaced with the him-5-deop to monitor the effect of CUB in mRNA expression. From the results of genotyping and phenotype observation, we selected the homozygous gfp knock-in edit for the second knock-in. Although genotyping results suggested gfp is knocked into the him-5 locus, sequencing results showed that the 3' gRNA site on the Him-5 locus was deleted, and the him-5 exon 1 was retained. This result suggests that it may not be able to use identical sites for consecutive CRISPR genome editing. Therefore, the PAM domain should be mutated after each knock-in to avoid Cas9 repeated cleavage. We are still trying to obtain the new knock-in strains. Although the model is yet established, it will become a novel tool for studying the role of CUB in mRNA expression levels in higher eukaryotes.

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