CRISPR-GE: A Convenient Software Toolkit for CRISPR-Based Genome Editing
CRISPR-GE：CRISPR 基因组编辑的便捷软件工具包

Dear Editor, 亲爱的编辑，

Use of the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (Cas9) and Cpf1 systems in plants (Ma et al., 2016; Wang et al., 2017) involves many steps, including the selection of appropriate specific target site(s) that should have no highly homologous sequences as the potential off-target sites in the genome, the design and synthesis of oligonucleotides involving the target sequences, the preparation of expression cassette(s) for the target single guide RNAs (sgRNAs) that provide target sequence specificity, the construction of plant-transformation/expression vector(s), and the transformation of plants, followed by the detection and determination of the targeted mutations in the transgenic plants. Several software tools have been developed for designing target sites or evaluating the outcome of genome/gene editing; however, to date, the toolkit that could aid a genome editing experiment simultaneously at all the steps is not available. Here we present a web-based software package, CRISPR-GE (Genome Editing) (http://skl.scau.edu.cn), a convenient, integrated toolkit to expedite all experimental designs and analyses of mutations for CRISPR/Cas9/Cpf1-based genome editing in plants and other organisms.
在植物中使用聚集的 CRISPR 相关蛋白 9（Cas9）和 Cpf1 系统（Ma 等，2016 年；Wang 等，2017 年）涉及许多步骤，包括选择适当的特定靶位点，这些位点不应在基因组中具有高度同源序列作为潜在的非靶位点，设计和合成涉及靶序列的寡核苷酸，为提供靶序列特异性的靶单导 RNA（sgRNA）准备表达载体，构建植物转化/表达载体，并转化植物，随后检测和确定转基因植物中的靶向突变。已开发了几种软件工具用于设计靶位点或评估基因组/基因编辑的结果；然而，迄今为止，尚无能够同时在所有步骤上协助基因组编辑实验的工具包。在这里，我们介绍一个基于网络的软件包，CRISPR-GE（基因组编辑）（http://skl.scau.edu.CRISPR-GE（CRISPR 基因组编辑）是一个方便、集成的工具包，用于加速植物和其他生物体中基于 CRISPR/Cas9/Cpf1 的基因组编辑的所有实验设计和突变分析。

CRISPR-GE provides a set of powerful tools for the design of target sgRNAs (targetDesign), prediction of off-target sites (offTarget), design of primers for construction of the sgRNA expression cassettes and amplification of target site-containing genomic fragments (primerDesign), determination of mutant sequences from sequencing chromatograms of genomic PCR amplicons containing target sites (DSDecodeM), and download of genomic sequences of certain regions from reference genomes (seqDownload). Using our previously reported CRISPR/ Cas9 vector system (Ma et al., 2015a) or other CRISPR/Cas9/ Cpf1 vector systems, CRISPR-GE offers an efficient and complete solution for genome editing in plants. Figure shows the overall workflow of CRISPR-GE.
CRISPR-GE 提供了一套强大的工具，用于设计目标 sgRNA（targetDesign）、预测非靶位点（offTarget）、设计引物以构建 sgRNA 表达载体和扩增含有目标位点的基因组片段（primerDesign）、从包含目标位点的基因组 PCR 扩增子的测序色谱图中确定突变序列（DSDecodeM），以及从参考基因组中下载特定区域的基因组序列（seqDownload）。使用我们先前报道的 CRISPR/Cas9 载体系统（Ma 等，2015a）或其他 CRISPR/Cas9/Cpf1 载体系统，CRISPR-GE 为植物基因组编辑提供了高效完整的解决方案。图 显示了 CRISPR-GE 的整体工作流程。

The first tool in CRISPR-GE, targetDesign, aids in the key step of choosing appropriate target site(s) for the Cas9 or Cpf1 nucleases. Moreover, the associated offTarget program facilitates the selection of specific target site(s) and prevents the cleavage at non-target sequences in the target genome. Using the targetDesign program, researchers can rapidly find all possible target sites in a given sequence of interest and make a prediction of potential off-target sites and estimated scores in the assigned genome by invoking the offTarget algorithm. Meanwhile, researchers can also select target sites with different types of protospacer-adjacent motif (PAM, including NGG for SpCas9, TTN for FnCpf1, TTTN for AsCpf1, or a specific PAM defined by the user) for the CRISPR systems. First, the user selects or defines the PAM type and a target/reference genome, and inputs a genomic sequence (up to ) of the target plant vari- ety/line, or a gene locus name, whereby the program will design the target site(s) (Figure 1B). If using a gene locus, the target gene sequence is derived from the selected target/reference genome. Currently, 27 plant genomes and genomes of other non-plant organisms, including human, mouse, zebrafish and Caenorhabditis elegans (Supplemental Table 1), are provided as the targets/ references. After submitting the task, targetDesign aligns the query sequence to the target/reference genome using BLASTN to locate its chromosomal positions. Then, the program (1) searches all possible target sites with the defined PAM within the forward and reverse strands, and (2) analyzes the secondary structure of the candidate target sgRNAs and indicates the target site(s) that have eight or more contiguous nucleotides to pair with the sgRNA scaffold sequence, which may affect the normal folding of the target sgRNA, thus reducing the editing efficiency (Ma et al., 2015a). Also, (3) targetDesign invokes the offTarget program to predict potential off-target sites in the target/reference genome and assign an estimated score to each potential off-target site of the CRISPR/ Cas9 system according to the previous analysis of the offtarget effects of each base in the target sgRNA in mammalian cells (Doench et al., 2016). Potential off-target sites with higher scores may have higher chances of being targeted by the sgRNA/Cas9 complex. After identifying potential off-target sequences, (4) targetDesign will query the annotation of each target site and off-target site. To quickly perform the procedure, we built and included a database containing annotations of the reference genomes in CRISPR-GE.
CRISPR-GE 中的第一个工具 targetDesign 有助于选择适当的 Cas9 或 Cpf1 核酸酶的靶位点这一关键步骤。此外，相关的 offTarget 程序有助于选择特定的靶位点，并防止在目标基因组中的非靶序列处发生切割。使用 targetDesign 程序，研究人员可以快速找到感兴趣序列中的所有可能靶位点，并通过调用 offTarget 算法在指定基因组中预测潜在的非靶位点和估计分数。同时，研究人员还可以为 CRISPR 系统选择具有不同类型的原始间质相邻基序（PAM，包括 NGG 用于 SpCas9，TTN 用于 FnCpf1，TTTN 用于 AsCpf1，或用户定义的特定 PAM）。首先，用户选择或定义 PAM 类型和一个靶位点/参考基因组，并输入靶植物品种/系列的基因组序列（最多 ），或基因座名称，程序将设计靶位点（图 1B）。如果使用基因座，靶基因序列源自所选的靶位点/参考基因组。 目前，提供了 27 个植物基因组和其他非植物生物的基因组，包括人类、小鼠、斑马鱼和秀丽隐杆线虫（附表 1），作为目标/参考。提交任务后，targetDesign 使用 BLASTN 将查询序列与目标/参考基因组进行比对，以定位其染色体位置。然后，该程序（1）搜索所有可能的目标位点，其中包含定义的 PAM 序列在正向和反向链上，（2）分析候选目标 sgRNA 的二级结构，并指示具有八个或更多连续核苷酸与 sgRNA 支架序列配对的目标位点，这可能会影响目标 sgRNA 的正常折叠，从而降低编辑效率（Ma 等，2015a）。此外，（3）targetDesign 调用 offTarget 程序，预测目标/参考基因组中的潜在离靶位点，并根据以前在哺乳动物细胞中对目标 sgRNA 的每个碱基的离靶效应进行的分析，为 CRISPR/Cas9 系统的每个潜在离靶位点分配一个估计分数（Doench 等，2016）。 潜在的得分较高的非靶位点可能更有可能被 sgRNA/Cas9 复合物靶向。在识别潜在的非靶序列后，（4）targetDesign 将查询每个靶位点和非靶位点的注释。为了快速执行该过程，我们构建并包含了一个包含 CRISPR-GE 参考基因组注释的数据库。

Moreover, (5) targetDesign can output the results in an interactive table (Figure 1C). The table lists all candidate target sites and their positions in the input genomic sequence, GC contents of the target sites, genomic locations, and corresponding potential off-target sites and their scores. All mismatched base(s) of the potential off-target sites to the target sgRNA are marked in red. Furthermore, restriction enzyme sites that are present in the candidate targets can be displayed by clicking the "Show Restriction Enzyme Sites" button. Since four or more contiguous " " bases in target sgRNAs may serve as a transcription termination signal for RNA polymerase III, which produces transcripts from the U3/U6 promoters, targetDesign marks such poly-T sites in candidate target sequences in yellow for attention. The detailed off-target information can be displayed by clicking the "see detail" link. The editing efficiency of target sites by CRISPR/Cas9/Cpf1 systems is related to the target sequence compositions, but also may be largely affected by other unknown factors, for example, the different chromatin states (e.g., euchromatin or heterochromatin, transcriptionally active or silencing) of the target site regions.
此外，（5）targetDesign 可以以交互式表格的形式输出结果（图 1C）。该表列出了所有候选靶点及其在输入基因组序列中的位置、靶点的 GC 含量、基因组位置以及相应的潜在非靶位点及其得分。潜在非靶位点与目标 sgRNA 的所有错配碱基均标记为红色。此外，点击“显示限制酶位点”按钮可以显示候选靶点中存在的限制酶位点。由于靶点 sgRNA 中存在四个或更多连续的“ ”碱基可能作为 RNA 聚合酶 III 的转录终止信号，从而产生 U3/U6 启动子的转录产物，targetDesign 会以黄色标记候选靶点序列中的这些多聚 T 位点以引起注意。点击“查看详情”链接可以显示详细的非靶信息。CRISPR/Cas9/Cpf1 系统对靶点的编辑效率与靶序列组成有关，但也可能受其他未知因素的影响，例如不同的染色质状态（例如。目标位点区域的染色质状态（如欧染质或异染质，转录活跃或沉默）。

Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.
由分子植物上海编辑办公室与 Cell Press 共同出版，Elsevier Inc.的一个品牌，代表 CSPB 和 IPPE、SIBS、CAS。

C Results of fob 10: 201706121941 smekL Results of targetDesign
C fob 10 的结果：201706121941 smekL targetDesign 的结果

Click Show Restricon Ename shes to show or hide restriction enzyme sines that are present in the candidate largets
单击“显示限制性酶位点”以显示或隐藏候选目标中存在的限制性酶位点

Figure 1. The CRISPR-GE Toolkit for Genome Editing.
图 1. CRISPR-GE 基因组编辑工具包。

(A) The overall workflow of CRISPR-GE.
(A) CRISPR-GE 的整体工作流程。

(B) The submission page of targetDesign.
(B) targetDesign 的提交页面。

(C) The results page of targetDesign and a table of results. Low-efficiency sites with contiguous "T" bases (four or more), very low or very high GC content ( or ), and contiguous base-pairing with the sgRNA sequence are indicated with "Bad site warning" marks (!, !!, and !!!). The mismatched bases in potential off-target sites are shown in red.
(C) targetDesign 的结果页面和结果表。具有连续“T”碱基（四个或更多）、非常低或非常高的 GC 含量（ 或 ）以及与 sgRNA 序列连续配对的低效位点，用“Bad site warning”标记（！，！！和！！！）表示。潜在的离靶位点中的错配碱基以红色显示。

(D) The submission page of offtarget.
(D) offtarget 的提交页面。

(E) Using primerDesign-V to generate primers for preparation of sgRNA expression cassette(s).
(E) 使用 primerDesign-V 生成引物，用于准备 sgRNA 表达载体。

(F) The results page of DSDecodeM. Multiple (up to 20) sequencing files (ab1 format) can be uploaded at the same time for decoding. All decoding results are displayed in the page and can be downloaded in a file (.txt) by clicking "here".
(F) DSDecodeM 的结果页面。最多可同时上传多个（最多 20 个）测序文件（ab1 格式）进行解码。所有解码结果都显示在页面上，并可通过单击“此处”下载到一个文件（.txt）。

Therefore, it may be difficult or impossible to precisely determine target sites of "high efficiency" for editing by the program based on the nucleotide compositions of the target sites. However, it is possible to anticipate the "low-efficiency" sites based on the common features that negatively affect the editing as described above. In summary, candidate sites with very low or very high GC content ( or , poly-T site(s), contiguous base-pairing with the sgRNA sequence, which are indicated by a "Bad site warning" mark (!, !!, or !!!), and those having potential off-target site(s) of high score values ( 0.7 or higher), if this issue is concerned, should not be used for the genome editing. Finally, (6) users can select appropriate target site(s) and clicks "Primer design" to enter primerDesign-V (Vector), a subprogram of primerDesign, to generate primers for the subsequent vector construction using our CRISPR/Cas9 vector system (Ma et al., 2015a), which has gained wide usage, or other CRISPR/Cas9 vectors.
因此，基于靶位点的核苷酸组成，可能很难或不可能精确确定程序编辑的“高效率”靶位点。然而，可以根据上述负面影响编辑的共同特征来预测“低效率”位点。总之，具有非常低或非常高 GC 含量（ 或 ，聚 T 位点，与 sgRNA 序列连续碱基配对，由“不良位点警告”标记（！，！！或!!!）指示的潜在离靶位点的高分值（0.7 或更高）的候选位点，如果涉及此问题，不应用于基因组编辑。最后，（6）用户可以选择适当的靶位点，并单击“引物设计”进入 primerDesign-V（Vector），primerDesign 的子程序，以使用我们的 CRISPR/Cas9 载体系统（Ma 等，2015a）生成用于后续载体构建的引物，该系统已被广泛使用，或其他 CRISPR/Cas9 载体。

Users also can directly enter the offTarget program to evaluate potential off-target sites of defined target site(s) in a target/reference genome. To perform this task, the user should select (or define) the PAM type and the target/reference genome, and input the target site sequence(s) and their corresponding PAM(s) (Figure 1D). The users can input multiple target sites by clicking the "Insert" button. After submitting the job, the query target site sequence(s) are aligned to the target/reference genome with mismatches of up to five nucleotides using BatMis algorithms (Tennakoon et al., 2012) , and the potential off-target sites for the CRISPR/Cas9 system are scored. Since no experimental analysis or reliable models for off-target effects of CRISPR editing systems using Cpf1 nucleases or other Cas9 variants are available, the program will skip the scoring step for off-target effects of these systems. The resulting table of the off-target predictor lists potential off-target sites with their scores, including the target sequence itself ( score ). The mismatched bases between the target sgRNAs and off-target sites are highlighted in red (Figure 1C).
用户还可以直接输入 offTarget 程序，评估目标/参考基因组中定义目标位点的潜在离靶位点。为执行此任务，用户应选择（或定义）PAM 类型和目标/参考基因组，并输入目标位点序列及其相应的 PAM（图 1D）。用户可以通过点击“插入”按钮输入多个目标位点。提交作业后，使用 BatMis 算法（Tennakoon 等，2012）将查询目标位点序列与目标/参考基因组进行对齐，允许最多五个核苷酸的错配，并对 CRISPR/Cas9 系统的潜在离靶位点进行评分。由于目前没有关于使用 Cpf1 核酸酶或其他 Cas9 变体的 CRISPR 编辑系统离靶效应的实验分析或可靠模型，该程序将跳过这些系统离靶效应的评分步骤。离靶预测器的结果表列出了潜在离靶位点及其得分，包括目标序列本身（得分 ）。目标 sgRNA 与离靶位点之间的错配碱基以红色突出显示（图 1C）。

Compared with other target-sgRNA design tools such as CRISPR-P (Liu et al., 2017), E-CRISPR (Heigwer et al., 2014), and Breaking-Cas (Oliveros et al., 2016), the targetDesign/ offTarget programs have the following advantages. First, targetDesign/offTarget allows users to design target sites (and predict potential off-target sites) in the inputted genomic sequences of the target varieties/lines that may actually have nucleotide variations with the selected reference genome (or the genome of the used close relative species), or in the gene sequences that are directly derived from the reference genome by the program (with the gene locus name). Second, if the reference genome sequence or the genome of a close relative species is not available, targetDesign also can be used to design target site(s) in the input genomic sequence with evaluations under the factors described above except for the prediction of potential off-target sites in the target/reference genome.
与其他目标-sgRNA 设计工具（如 CRISPR-P（Liu 等，2017 年），E-CRISPR（Heigwer 等，2014 年）和 Breaking-Cas（Oliveros 等，2016 年））相比，targetDesign/offTarget 程序具有以下优势。首先，targetDesign/offTarget 允许用户在目标品种/系的输入基因组序列中设计目标位点（并预测潜在的非靶位点），这些位点可能实际上与所选参考基因组（或所用近缘物种的基因组）具有核苷酸变异，或者在程序直接从参考基因组中派生的基因序列中（带有基因位点名称）。其次，如果参考基因组序列或近缘物种的基因组不可用，targetDesign 也可用于在输入基因组序列中设计目标位点，并在上述因素下进行评估，但不包括在目标/参考基因组中预测潜在的非靶位点。

To prepare CRISPR/Cas9/sgRNA constructs, the first step is to generate target-sgRNA expression cassette(s). The target site(s) selected through targetDesign and/or offTarget are transferred to primerDesign-V, which automatically outputs the primers for preparation of the sgRNA expression cassettes. Users also can directly enter primerDesign/primerDesign-V for the primer generation. Our CRISPR/Cas9/sgRNA vector system provides four U3 and U6 small nuclear RNA promoters from rice and four U3 and U6 promoters from Arabidopsis to express the target-sgRNAs in plants. Two methods can be used to prepare the target-sgRNA expression cassettes: Adapter-ligation/PCR (Method 1), or Overlapping PCR (Method 2) (Ma et al., 2015a; Ma and Liu, 2016). Depending on the selected promoter(s) and method, different primers are generated to introduce the target sequences into the target-sgRNA expression cassettes (Figure 1E). The primerDesign-V tool also can be used to generate the adapter primers for other sgRNA vector systems that use the Adapterligation method.
为准备 CRISPR/Cas9/sgRNA 构建，第一步是生成目标-sgRNA 表达载体。通过 targetDesign 和/或 offTarget 选择的目标位点被转移到 primerDesign-V，该工具会自动生成用于准备 sgRNA 表达载体的引物。用户也可以直接输入 primerDesign/primerDesign-V 来生成引物。我们的 CRISPR/Cas9/sgRNA 载体系统提供了来自水稻的四个 U3 和 U6 小核 RNA 启动子以及来自拟南芥的四个 U3 和 U6 启动子，用于在植物中表达目标-sgRNA。有两种方法可用于准备目标-sgRNA 表达载体：适配子连接/PCR（方法 1）或重叠 PCR（方法 2）（Ma 等，2015a；Ma 和 Liu，2016）。根据所选的启动子和方法，会生成不同的引物，将目标序列引入目标-sgRNA 表达载体（图 1E）。primerDesign-V 工具还可用于生成适配子引物，用于其他使用适配子连接方法的 sgRNA 载体系统。

To facilitate PCR-amplification of genomic sequences that contain one or multiple target sites for analysis of the targeted mutations, we included another subprogram, primerDesign-A (Amplification), in primerDesign. With this tool, highly specific primers for the genomic PCR, and those for consequent sequencing, are designed by analyzing the reference genome sequence using the Primer3 (Untergasser et al., 2012) and BatMis algorithms (Tennakoon et al., 2012). The locations of the target site(s), the selected primers, and the sequencing primer(s) are displayed directly in the genomic sequence. The amplicons using these primers can be used for direct sequencing followed by decoding using the tool described below, for cloning in plasmid vectors or for polymorphism detection by other methods.
为了便于 PCR 扩增包含一个或多个目标位点的基因组序列，以便分析目标突变，我们在 primerDesign 中包含了另一个子程序 primerDesign-A（扩增）。使用这个工具，通过分析参考基因组序列，使用 Primer3（Untergasser 等，2012 年）和 BatMis 算法（Tennakoon 等，2012 年），设计用于基因组 PCR 和随后测序的高度特异性引物。目标位点的位置、选择的引物以及测序引物直接显示在基因组序列中。使用这些引物的扩增子可以用于直接测序，随后使用下面描述的工具进行解码，用于克隆到质粒载体或通过其他方法进行多态性检测。

For analysis of transgenic plants obtained with the editing constructs, CRISPR-GE provides the DSDecodeM program, an updated version of DSDecode (Liu et al., 2015), to determine the allelic mutant sequences of targeted sites. In diploid organisms, CRISPR-mediated genome editing can often cause uniform (biallelic, heterozygous, and homozygous) mutations. Direct sequencing of PCR amplicons containing such biallelic and heterozygous mutant sites produces superimposed sequencing chromatograms. Previously we developed the DSD (Degenerate Sequence Decoding) method and its web-based software tool (DSDecode) for decoding allelic mutant sequences of various uniform mutations from sequencing chromatograms with superimposed peaks (Liu et al., 2015; Ma et al., 2015b). However, DSDecode can only analyze one sequencing chromatogram file (ab1 format) at a time. Here, the updated DSDecodeM can analyze up to 20 sequencing chromatograms at the same time. In addition, DSDecodeM has several other improvements compared with DSDecode. (1) If higher noise signals are produced in the region before the target sites of sequencing chromatograms, the noise may interfere with the analysis of the program, leading to failure of the decoding. In DSDecodeM, the target sequence can be provided, as an optional input, which may allow the program to exclude these noise signals, thus enabling correct decoding. (2) To make the program more flexible in separating sequencing signal from noise, DSDecodeM has an optional parameter setting to adjust the cutoff of the noise-peak/base-peak signal ratio if using the default ratio (0.3) fails to decode sequencing chromatograms of lower qualities. (3) DSDecode reads the sequencing digital information from ab1 files using the sangerseqR package, but this consumes more computational resources and thus requires a relatively long time for the decoding. In DSDecodeM we developed a Python-based program that can rapidly read the digital information from ab1 files. Therefore, the decoding speed of DSDecodeM is much faster ( per ab1 file if multiple files are decoded at the same time) than that of DSDecode ( per ab1 file). (4) The result page of DSDecodeM displays all decoding results of multiple sequencing files, which can be downloaded in a txt-format file (Figure 1F).
对于使用编辑构建获得的转基因植物的分析，CRISPR-GE 提供 DSDecodeM 程序，这是 DSDecode（Liu 等，2015 年）的更新版本，用于确定靶位点的等位突变序列。在二倍体生物中，CRISPR 介导的基因组编辑通常会导致统一的（双等位、杂合和纯合）突变。直接测序包含这种双等位和杂合突变位点的 PCR 扩增子会产生叠加的测序色谱图。我们先前开发了 DSD（退化序列解码）方法及其基于网络的软件工具（DSDecode），用于从具有叠加峰的测序色谱图中解码各种统一突变的等位突变序列（Liu 等，2015 年；Ma 等，2015b 年）。然而，DSDecode 一次只能分析一个测序色谱图文件（ab1 格式）。在这里，更新的 DSDecodeM 可以同时分析多达 20 个测序色谱图。此外，与 DSDecode 相比，DSDecodeM 还有几项改进。 （1）如果在测序色谱图的目标位点之前的区域产生了更高的噪音信号，这些噪音可能会干扰程序的分析，导致解码失败。在 DSDecodeM 中，可以提供目标序列作为可选输入，这可能使程序排除这些噪音信号，从而实现正确的解码。（2）为了使程序在分离测序信号和噪音方面更加灵活，DSDecodeM 具有一个可选参数设置，用于调整噪音峰值/基础峰值信号比的截止值 ，如果使用默认比值（0.3）无法解码质量较低的测序色谱图。（3）DSDecode 使用 sangerseqR 软件包从 ab1 文件中读取测序数字信息，但这会消耗更多的计算资源，因此需要相对较长的时间进行解码。在 DSDecodeM 中，我们开发了一个基于 Python 的程序，可以快速从 ab1 文件中读取数字信息。因此，DSDecodeM 的解码速度比 DSDecode 快得多（ 每个 ab1 文件，如果同时解码多个文件）。 (4) DSDecodeM 的结果页面显示了多个测序文件的所有解码结果，可以下载为 txt 格式文件（图 1F）。

The tool seqDownload is convenient for downloading genomic sequences of various lengths (up to ca. ) of certain regions from the selected reference genomes (Supplemental Table 1), for various analyses. For this sequence download, a user needs to input a gene locus name, a short marker sequence, or a pair of marker primer sequences located on the target region, and to define the lengths of the upstream and downstream flanking sequences to the gene locus/marker sequence.
工具 seqDownload 方便用户从选定的参考基因组（附录表 1）中下载各种长度（最长约 ）的基因组序列，用于各种分析。对于这种序列下载，用户需要输入基因座名称、短标记序列或位于目标区域上游和下游的标记引物序列，并定义上游和下游的序列长度到基因座/标记序列。

In summary, the overarching purpose of CRISPR-GE is to establish an intuitive and powerful toolkit for researchers to perform
总的来说，CRISPR-GE 的主要目的是建立一个直观而强大的工具包，供研究人员进行操作。
genome editing. This toolkit will greatly facilitate the entire design and analysis process, enabling high-efficiency CRISPR-based genome editing in plants and other organisms.
基因组编辑。这个工具包将极大地促进整个设计和分析过程，实现在植物和其他生物中高效的 CRISPR 基因组编辑。

Supplemental Information is available at Molecular Plant Online.
补充信息可在《分子植物在线》获取。

This work was supported by grants from Guangdong Province Public Interest Research and Capacity Building Special Fund (2015B020201002), the Ministry of Agriculture of the People's Republic of China (2016ZX08010-001, 2016ZX08009-002), and the Postdoctoral Science Foundation of China (2016M602480).
本工作得到广东省公益研究与能力建设专项资金（2015B020201002）、中华人民共和国农业部（2016ZX08010-001，2016ZX08009-002）以及中国博士后科学基金（2016M602480）的资助。

X.X. wrote, modified, and tested the programs, and wrote the paper; X.M., Q.Z., and D.Z. tested the programs; G.L. designed the homepage of DSDecodeM; and Y.-G.L. supervised the project, designed the program structure, and wrote the paper.
X.X.编写、修改和测试程序，并撰写论文；X.M.、Q.Z.和 D.Z.测试程序；G.L.设计了 DSDecodeM 的主页；Y.-G.L.监督项目，设计程序结构，并撰写论文。

No conflict of interest declared.
未声明利益冲突。

Received: April 18, 2017
收到日期：2017 年 4 月 18 日

Revised: June 5, 2017
修订日期：2017 年 6 月 5 日

Accepted: June 11, 2017
接受日期：2017 年 6 月 11 日

Published: June 14, 2017
发布日期：2017 年 6 月 14 日

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangzhou 510642, China Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions, Guangzhou 510642, China
中国广州 510642 亚热带农业生物资源保护与利用国家重点实验室 广东省高校植物功能基因组学与生物技术重点实验室，中国广州 510642

College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
中国华南农业大学生命科学学院，中国广州 510642

Present address: Global Institute for Food Security, University of Saskatchewan, Saskatoon, SK S7N 5A2, Canada Correspondence: Yao-Guang Liu (ygliu@scau.edu.cn) http://dx.doi.org/10.1016/j.molp.2017.06.004
现地址：加拿大萨斯喀彻温省萨斯卡通，萨斯卡通大学全球食品安全研究所 通讯：刘耀光（ygliu@scau.edu.cn）http://dx.doi.org/10.1016/j.molp.2017.06.004

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