1. Background

The CRISPR/Cas9 technology is a modern, fashionable method in plant research. Immediately after its early use to edit the genomes of animals and bacteria, its efficacy was demonstrated in the model plant systems of Arabidopsis, rice, sorghum, and tobacco. Nowadays, this technology is broadly used in different plant species, and dozens of CRISPR/Cas9 vectors are available in the public plasmid repository of Addgene. The CRISPR/Cas9 pioneers have improved this system into a flexible and powerful platform for genome engineering. In this review, we briefly summarize the recent advances of CRISPR/Cas9 technology and its impact for plant genome engineering. More information could access in the review article (Ding et al., 2016): "Recent Advances in Genome Editing Using CRISPR/Cas9" in Front. Plant Sci.

We collected some of the key events in the development of CRISPR, click to the details.

The major concern about this technology is that Cas9 has off-target effects. Recent studies also showed that different guide sequences in sgRNAs have variable efficiency in genome editing (Doench et al., 2016; Fu et al., 2014; Liang et al., 2016). Therefore, the choice of targeting sites (same as the guide sequence of sgRNA) is the critical step in CRISPR-Cas9 technology.

2. Quick Guide for CRISPR-P 2.0 Users

The following list provides a swift overview of the steps involved in sgRNA design (Figure 1). Note: Please read the step by step guide to prevent frustration and to ensure optimal results.

  1. Please access "Submit" page to start a job. Select a genome you are studying, input gene locus, chromosome position, or DNA sequence in FASTA format. Other parameters such as PAM, snoRNA promoter, RNA scaffold, guide sequence (spacer) length are also need be defined or with default arguments. Then, click "Submit" button. If your sequence could not be found in the blast search of your selected genome, you can jump to 4
  2. A few seconds after submission, the web browser would automatically enter the Result page of sgRNA design. Firstly, the target sequence is mapped to its genome. All possible sgRNA are screened out and showed in graphic genome model and information about potential on-target and off-target sits are displayed, including: on-target score, off-target score, GC content, restriction endonuclease site, etc.
  3. Clicking "advance" button to enter the "Advanced result page of sgRNA design". It provided secondary structure of sgRNA and microhomology score for processes a further chose of efficient sgRNA. Designing sgRNAs according to your needs.
  4. User can identify on-target score from custom sgRNA sequence in "Design" page
Figure 1. The overall procedure of sgRNA guide design using CRISPR-P 2.0

Figure 1. The overall procedure of sgRNA guide design using CRISPR-P 2.0

3. Optimal sgRNA Design Step by Step in a Preset Genome

This section describes how to use the Optimized CRISPR-Plant 2.0 Design tool step by step in preset 49 plants. The following displays a detailed procedure for optimal sgRNA design.

4. Identifying On-target Efficiency Score from Custom sequence

User can identify On-target Efficiency Score from custom sgRNA sequences in a standalone sgRNA design module. This module has two basic functions:

  1. Estimating On-target Efficiency Score from custom sgRNAs;
  2. Identifying sgRNAs from custom long squences and then estimate thise sgRNAs with on-target score;

In page of "design" user can custom sgRNA multi-sequences in FASTA that more than 30 nt (included PAM "NGG"): Custom sgRNA sequence in format "NNNN + spacer (20bp) + NGG + NNN" or long sequences containing one or more sgRNA sequence of the format. Input content exampled in text as followed, the result of on-target score of target sequences are list in Figure 6.

Figure 6. Result of on-target score from custom sgRNA sequences

Figure 6. Result of on-target score from custom sgRNA sequences

5. Design of Score System

Design of On-target Score and Off-target Score system.

6. Contact

Project Leader: Lingling Chen, Kabin Xie
Any question contact us