Class2 CRISPR-Cas系统发掘及分析方法

Methods for Discovery and Analysis of Class2 CRISPR-Cas Systems

近年来,规律间隔成簇短回文系统(CRISPR-Cas)作为基因编辑手段在动植物基因编辑中已广泛应用。现已被证实的Class2类CRISPR-Cas系统CRISPR-Cas12、CRISPR-Cas14等均通过生物信息学手段被发掘出来,因此,生物信息学成为发现新CRISPR-Cas系统及其子类型的重要方法。笔者综述了Cas酶两类生物信息学发掘手段,一类方法是通过已知Cas酶建立隐马尔科夫模型(HMM)预测可能的同类Cas酶;另一类方法是以标志序列Cas1或CRISPR识别为基础分析上下游可能的Cas酶,同时讨论了两种方法的限制。在此基础上,综述了Cas蛋白和CRISPR序列进一步分析方法,包括Cas蛋白同源性、进化分析及CRISPR序列间隔序列(spacers)、前间隔序列(protospacers)前间隔序列临近基序(PAM)分析。

Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR-Cas)has been widely used as a tool in recent years for gene editing in animal and plant gene editing.The proven Class2 CRISPR-Cas systems,such as CRISPR-Cas12 and CRISPR-Cas14,have been discovered through bioinformatics mining.Bioinformatics has become an important tool for discovering of new CRISPR-Cas systems and their subtypes.Two methods for bioinformatics mining of Cas enzymes are reviewed.One method is to create a hidden Markov model(HMM)using known Cas enzymes to predict similar Cas enzymes,and the other method is to analyze the possible upstream and downstream Cas enzymesbased on the recognition of the marker sequence Cas1 or CRISPR.The limitations of these two methods are discussed.Furthermore,methods for further analysis of Cas protein and CRISPR sequences are also reviewed,including Cas protein homology,phylogenetic analysis,and analysis of CRISPR sequence spacers,protospacers&protospacer adjacent motifs(PAM).