摘要：
摘要：
Cas9-based technologies rely on native elements of Type II CRISPR–Cas bacterial immune systems, including the trans-activating CRISPR RNA (tracrRNA), CRISPR RNA (crRNA), Cas9 protein, and protospacer-adjacent motif (PAM). The tracrRNA and crRNA form an RNA duplex that guides the Cas9 endonuclease to complementary nucleic acid sequences. Mechanistically, Cas9 initiates interactions by binding to the target PAM sequence and interrogating the target DNA in a 3′ -to-5′ manner. Complementarity between the guide RNA and the target DNA is key. In natural systems, precise cleavage occurs when the target DNA sequence contains a PAM flanking a sequence homologous to the crRNA spacer sequence. Currently, the majority of commercial Cas9-based genome-editing tools are derived from the Type II CRISPR–Cas system of Streptococcus pyogenes. However, a diverse set of Type II CRISPR–Cas systems exist in nature that are potentially valuable for genome engineering applications. Exploitation of these systems requires prediction and validation of both native and engineered dual and single guide RNAs to drive Cas9 functionality. Here, we discuss how to identify the elements of these immune systems to develop next-generation Cas9-based genome-editing tools. We first discuss how to predict tracrRNA sequences and suggest a method for designing single guide RNAs containing only critical structural modules. We then outline how to predict the PAM sequence, which is crucial for determining potential targets for Cas9. Finally, validation of the system elements through transcriptome analysis and interference assays is essential for developing next-generation Cas9-based genome-editing tools.
要点：
Cas9技术需要：II型 CRISPR–Cas 细菌免疫系统，包括 trans-activating CRISPR RNA (tracrRNA), CRISPR RNA (crRNA), Cas9 protein, and protospacer-adjacent motif (PAM)。
trans-activating CRISPR RNA (tracrRNA), CRISPR RNA (crRNA)作用：形成RNA双链，引导Cas9核酸内切酶和核酸序列配对。
Cas9和目标PAM序列连接。
关键点：guide RNA 和目标DNA的互补。
大多数商业Cas9基因编辑工具来源：Streptococcus pyogenes（链球菌）的II型 CRISPR–Cas 系统。不过自然界还存在很多具有基因编辑潜力的II型 CRISPR–Cas 系统。开发这种系统需要预测验证天然或基因工程来源的，驱动Cas9的单双链RNA。
本文内容：
预测和设计tracrRNA序列。
预测PAM序列。
转录组分析法验证系统。

试剂：
Direct-zol RNA MiniPrep Kit (Zymo Research)
The Direct-zol RNA MiniPrep Kit from Zymo Research is highly recommended for extraction of RNA as it allows purification of RNA molecules as small as 17 nt, thereby retaining the smaller crRNAs that many traditional kits discard.
Next-generation sequencing reagents (HiSeq or MiSeq; Illumina)
Sample for RNA extraction
TruSeq Small RNA Sample Preparation Kit (Illumina)
要点：
RNA提取纯化试剂盒：适合小RNA，最低17碱基。
Illumina NGS基因测序试剂盒
RNA提取样本
Illumina TruSeq小RNA文库准备试剂盒

分析工具
Basic Local Alignment Search Tool (BLAST) and Conserved Domain Database (CCD) (National
Center for Biotechnology Information [NCBI])
CRISPR identification program
CRISPRfinder (http://crispr.u-psud.fr/Server/) is a web-based CRISPR identification platform that allows users to upload their own sequences of interest as well as browse genomes annotated directly from the NCBI database (Grissa et al. 2007). CRISPR Recognition Tool (CRT) (http://www.room220.com/crt/ [Bland et al. 2007]) is available as a command-line tool or plug-in for several commercially available graphical user interface (GUI) bioinformatics platforms (e.g., Geneious9 [Biomatters]). Both programs identify potential CRISPR spacers and repeats, based on a repeat-finding algorithm.
Genome sequence of interest
Quality control software for RNA sequencing data (e.g., FastQC)
RNA folding prediction software (e.g., NUPACK; http://www.nupack.org/ [Zadeh et al. 2011])
Sequence motif identification program (e.g., WebLogo [Crooks et al. 2004])
Short read sequence alignment tool (e.g., Bowtie 2 [Langmead and Salzberg 2012])
要点：
CRISPR识别软件：基于重复寻找算法，识别CRISPR的间隔和重复。
目标基因序列
RNA测序数据质量控制软件
RNA折叠预测软件
序列功能域识别程序
短序列比对工具