Data_Sheet_1_The Codon Usage Bias Analysis of Free-Living Ciliates’ Macronuclear Genomes and Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Vector Construction of Stylonychia lemnae.DOCX

Ciliates represent higher unicellular animals, and several species are also important model organisms for molecular biology research. Analyses of codon usage bias (CUB) of the macronuclear (MAC) genome in ciliates can not only promote a better understanding of the genetic mode and evolution history of these organisms but also help optimize codons to improve the gene editing efficiency of model ciliates. In this study, macronuclear genome sequences of nine free-living ciliates were analyzed with CodonW software to calculate the following indices: the guanine-cytosine content (GC); the frequency of the nucleotides U, C, A, and G at the third position of codons (U3s, C3s, A3s, G3s); the effective number of codons (ENC); the correlation between GC at the first and second positions (GC12); the frequency of the nucleotides G + C at the third position of synonymous codons (GC3s); the relative synonymous codon usage (RSCU). Parity rule 2 plot analysis, neutrality plot analysis, and correlation analysis were performed to explore the factors that influence codon preference. The results showed that the GC contents in nine ciliates’ MAC genomes were lower than 50% and appeared AT-rich. The base compositions of GC12 and GC3s are markedly distinct and the codon usage pattern and evolution of ciliates are affected by genetic mutation and natural selection. According to the synonymous codon analysis, the codons of most ciliates ended with A or U and eight codons were the general optimal codons of nine ciliates. A clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) expression vector of Stylonychia lemnae was constructed by optimizing the macronuclear genome codon and was successfully used to knock out the Adss gene. This is the first such extensive investigation of the MAC genome CUB of ciliates and the initial successful application of the CRISPR/Cas9 technique in free-living ciliates.

纤毛虫（Ciliates）属于高等单细胞动物，多个物种亦是分子生物学研究的重要模式生物。对纤毛虫大核（macronuclear, MAC）基因组的密码子使用偏好性（codon usage bias, CUB）开展分析，不仅可加深对这类生物遗传模式与进化历程的理解，还能辅助优化密码子以提升模式纤毛虫的基因编辑效率。本研究采用CodonW软件，对9种自由生活纤毛虫的大核基因组序列进行分析，计算得到如下指标：鸟嘌呤-胞嘧啶含量（guanine-cytosine content, GC）、密码子第三位核苷酸U、C、A、G的出现频率（U3s、C3s、A3s、G3s）、有效密码子数（effective number of codons, ENC）、第一、二位密码子的GC含量相关性（GC12）、同义密码子第三位的G+C含量（GC3s）以及相对同义密码子使用频率（relative synonymous codon usage, RSCU）。通过二规则奇偶图分析、中性图分析与相关性分析，本研究探究了影响纤毛虫密码子偏好性的相关因素。结果表明，9种纤毛虫大核基因组的GC含量均低于50%，呈现AT偏好特性。GC12与GC3s的碱基组成存在显著差异，纤毛虫的密码子使用模式与进化过程同时受到遗传突变与自然选择的共同影响。基于同义密码子分析结果，多数纤毛虫的密码子以A或U结尾，且8种密码子为9种纤毛虫共有的最优密码子。本研究通过优化莱氏棘尾虫（Stylonychia lemnae）的大核基因组密码子，构建了成簇规律间隔短回文重复序列/Cas9（clustered regularly interspaced short palindromic repeats/Cas9, CRISPR/Cas9）表达载体，并成功实现了Adss基因的敲除。本研究不仅是首次针对纤毛虫大核基因组密码子使用偏好性开展的大规模系统性研究，同时也是自由生活纤毛虫中CRISPR/Cas9技术的首次成功应用。