Document for using CRISPR-GE to perform genome editing

OUR FOCUS:

We focus on the development of vector systems and convenient software for researchers to perform CRISPR-based genome editing.



Attention:

(Please use IE9 or a higher version of Internet Explorer (IE10, IE11), or "Firefox", "Chrome" to perform the software)

If you are using "IE7"、"IE8" or "360 browser", it will cause failure to display the results of CRISPR-GE.



1. Workflow of CRISPR-GE

CRISPR-GE (Genome Editing) provides a set of 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 genomic DNA containing target site(s) (primerDesign), determination of mutant sequences from complex sequencing chromatograms of PCR amplicons (DSDecodeM), and download of genomic sequences of certain regions from reference genomes (seqDownload). With the assistance of our previously reported CRISPR/Cas9 vector system, CRISPR-GE offers an efficient solution for plant genome editing.

The overall workflow of CRISPR-GE


2. Quick Guidance for targetDesign

The targetDesign is a quick guide-RNA designer for CRISPR/Cas9/Cpf1 genome editing in plants. To start a new task, please follow the steps below:

Step 1 - define your PAM type and genome: The program provides three PAM type to select (NGG, TTN, TTTN). If you want to define another PAM instead, please select "User defined", and input your own PAM parameters in the text box.

Step 2 - select the target genome: If your target gene has an available genome (or close relative genome), please select the corresponding genome for off-target prediction, otherwise, you can select "None", without the prediction of potential off-target.

Step 3 - input the sequence of your target gene or fragment (genomic sequence): before entering the sequence or gene locus, please confirm you have selected corresponding target genome. If your input is a gene locus, you can change the upstream and downstream length.

Step 4 - submit the job: click the "Submit" button to start the program.

Results of targetDesign:

Column 1: select the suitable target site(s) to enter primerDesign for primer generation.

Column 2: bad site warning' marks. Targets that have polyT, very low or very high GC content (= < 25% or = > 80%), and contiguous base-pairing with the sgRNA sequence are indicated with ‘Bad site warning’ marks (!, !!, and !!!).

Column 3: list all possible target sequence, contiguous “T” bases (>=4) in target sequences are marked with yellow for attention

Column 4: the position of target sequence in the giving sequence.

Column 5: the strand of target sequence in the giving sequence. ("-" or "+")

Column 6: GC content (%) of the target sequence

Column 7: the chromosomal region of the target sequence in the genome. If no target genome selected, the region are displayed as "NA".

Column 8: corresponding potential off-target sites and their scores. click the "see detail" to display detail information.

Column 9: target-sgRNAs pairing. Target site(s) that have eight or more contiguous nucleotides to pair with the sgRNA sequence may have low editing efficiency.

Click button can show the restriction enzyme site present in the candidate targets.

Optional step - go to primer design: click the "Primer Design" button to enter primerDesign-V to generate primers for vector construction if using our CRISPR/Cas9 vector system or other sgRNA vector systems that use the Adapter-ligation method.



3. Quick Guidance for offTarget

The offTarget is designed for predicting the potential off-target sites of Cas9/Cpf1 RNA-guided endonucleases.

Step 1 - define your PAM type: The program provides three PAM type to select (NGG, TTN, TTTN). If you want to define another PAM instead, please select "User defined", and input your own PAM parameters in the text box.

Step 2 - select the target genome: select the genome of your targeted species.

Step 3 - input the target site sequence(s) and their corresponding PAM(s): Users can input multiple target sites by clicking the “Insert” button.

Step 4 - submit the job: click the "Submit" button to start the program.

Results of offTarget:

The mismatched bases in potential off-target sites are indicated in red. Potential off-target sites with higher scores may have higher off-target risks of being targeted by the sgRNA/Cas9 nuclease complex.

Note: The off-target scoring based on reference: Doench JG, Fusi N, Sullender M, et al., Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. 2016, Nat. Biotechnol., 34(2):184-91.


4. Quick Guidance for primerDesign

Currently, primerDesign includes two subprogram: primerDesign-V and primerDesign-A.

primerDesign-V: design primers for construction of the sgRNA expression cassettes.

primerDesign-A: design specific PCR primers for amplification of target site-containing genomic fragments.


(1) primerDesign-V: automatically generates primers for vectors constructing of CRISPR/Cas9 genome editing in plants.

For vector system based on Liu YG Lab, pleas enter your inputs as following figure.

Step 1 - Input the target sequence (20 nt): please confirm that the direction of sequence is 5' to 3'.

Step 2 - select the promoter

Step 3 - select the method to prepare the target-sgRNA expression cassettes: method1: adapter-ligation and PCR; method2: overlapping PCR.

Step 4 - design the primers: click the "Design" button to design the primers.

For other sgRNA vector systems that use the Adapter-ligation method, pleas enter your inputs as following figure.

Step 1 - Input the target sequence (19 or 20 nt): please confirm that the direction of sequence is 5' to 3'.

Step 2 - Enter the 4 nucleotides (bold red) as shown in the Figure.

Step 3 - If the used sgRNA vector differs from the default one (bold blue), change it accordingly.

Step 4 - design the primers: click the "Design" button to design the primers.


(2) primerDesign-A: design specific PCR primers for amplification of target site-containing genomic fragments.

Step 1 - select the reference genome

Step 2 - input your PCR template: your template can be a sequence or a gene locus

Step 3 - target site setting: set your target sequence and the primers position.

The result table shows specific PCR primers for amplification. The sequence information can be displayed by clicking the ID.



5. Quick Guidance for seqDownload

seqDownload: A convenient tool to download genomic sequence using a seed sequence or gene.

The tool is used for users to download genomic sequences of various lengths of certain regions from the selected reference genomes.

Step 1 - select the reference genome

Step 2 - input a gene locus, or a marker sequence, or a pair of primer sequences

Step 3 - defines the lengths of the upstream and downstream sequences to the gene locus/seed sequence.

The result highlights the inputted marker/gene sequence with green background color.



6. Quick Guidance for DSDecodeM

DSDecodeM: An updated web tool for rapid decoding of multiple superimposed sequencing chromatograms

The DSDecodeM software serves to decode automatically sequencing chromatograms with biallelic, heterozygous, and homozygous mutations into allelic sequences.

The noise signal will interfere with the analysis of the program. To obtain a higher quality sequencing file, we suggest users to prepare the PCR fragment as following:

An examples to detect the mutation of one target site contained:

To decode two (or more) target sites in an amplicon, we suggest to design the primers as follows (each target site uses a sequencing primer):


After obtaining the sequencing file(s), please follow steps below to decode the mutation:

Step 1 - Input the reference sequence: enter an intact (wild-type) reference sequence. It must completely cover the sequence range of the sequencing ab1 file(s).

Step 2 - Upload chromatogram files: multiple ab1 files (up to 20) can be uploaded at the same time. It's better to check the sequencing quality of the ab1 files before decoding.

Step 3 - set optional parameters: usually it is not needed to change the default settings and enter a target sequence.

Step 4 - decode: click the "Decode" button to design the primers.



Project Manager: Yao-Guang Liu, Xianrong Xie

Contact:

E-mail: skl-cusa@scau.edu.cn



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