User Guide to gBIG

gRNA design process on gBIG

Inputs

gBIG supports DNA sequence inputs in the form FASTA files. Space and "_" should be avoided in the target gene name.

Example for target gene:

>Cgl0413

ATGGGTTCTGTCATCAAGAAGCGCCGCAAGCGCATGTCCAAGAAGAAGCACCGCAAGATGCTGCGCCGTACTCGTGTCCAGCGTAGAAAATTGGGCAAGTA...

Example for reference genome:

>Corynebacterium_glutamicum_ATCC13032

GTGAGCCAGAACTCATCTTCTTTGCTCGAAACCTGGCGCCAAGTTGTTGCCGATCTCACAACTTTGAGCCAGCAAGCGGACAGTGGATTCGACCCATTGAC...

Outputs

There are 31 columns in the output file "sgRNA_result_editable.csv"

Item Info
sgRNA_ID ID for each sgRNA
Start The start position of sgRNA in the target gene
End The end position of sgRNA in the target gene
CRISPR_target_sequence (5'-3') CRISRP target sequence (5'-3'), containing PAM
sgRNA_Sequence sgRNA sequence (5'-3')
PAM_Sequence PAM sequence
CRISPR_target_sequence_Length (nt) Length of CRISPR target sequence containing PAM (nt)
GC% GC content of CRISPR target sequence
1st_target_codon The first target codon
Editable_for_forming_an_early_stop_codon "1" represents this codon can be edited to form an early stop codon. "0" represents this codon can be edited but no early stop codon will be generated
Position_of_C_inside_gRNA Position of the target C inside the gRNA
Position_of_C_inside_the_gene (%) Position of the target C inside the gene (%)
2nd_target_codon The second target codon
Editable_for_forming_an_early_stop_codon "1" represents this codon can be edited to form an early stop codon. "0" represents this codon can be edited but no early stop codon will be generated
Position_of_C_inside_gRNA Position of the target C inside the gRNA
Position_of_C_inside_the_gene (%) Position of the target C inside the gene (%)
M0_OT On-target site. 0M_OT>1 represents repeat genes or off-target sites with 0 mismatch
M1_OT No. of off-target sites with 1 mismatch
M2_OT No. of off-target sites with 2 mismatches
M3_OT No. of off-target sites with 3 mismatches
M4_OT No. of off-target sites with 4 mismatches
M5_OT No. of off-target sites with 5 mismatches
Total_No.of_OT Total No. of off-target sites
M0_POT Perfect match to the 12-nt seed region of the sgRNA and 0 mismatch in the outer segments
M1_POT Perfect match to the 12-nt seed region of the sgRNA and 1 mismatch in the outer segments
M2_POT Perfect match to the 12-nt seed region of the sgRNA and 2 mismatches in the outer segments
M3_POT Perfect match to the 12-nt seed region of the sgRNA and 3 mismatches in the outer segments
M4_POT Perfect match to the 12-nt seed region of the sgRNA and 4 mismatches in the outer segments
M5_POT Perfect match to the 12-nt seed region of the sgRNA and 5 mismatches in the outer segments
Total_No.of_POT Total number of off-targets sites with perfect match to the 12-nt seed region of the sgRNA
Risk_evaluation Evaluation of off-target risk

Notes: OT stands for off-targets, which contain all types of off-target sites; POT stands for potential off-target, only contain off-targets which are perfect match to the 12-nt seed region of the guide RNA, while outer segments contain mismatched bases.

Off-target risk of guide RNA:

Discard > High_risk > moderate_risk> low_risk > repeat_sites_or_bad > Best

Notes: gBIG is free for academic, nonprofit, and personal use.

If you have any questions, please contact us: yangyi@tib.cas.cn.