Online tool for designing gRNA for Base editor-mediated Inactivation of Genes (gBIG)

gBIG designs guide RNAs for base editing-mediated inactivation of given genes in a given sequenced genome.

PAM Type

CRISPR/Cas variant

Base Editor Type

1 Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, Mochizuki M, Miyabe A, Araki M, Hara KY, Shimatani Z, Kondo A. 2016. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353:aaf8729.

2 Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. 2016. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533:420–424.

Reference Genome

Chose

Select reference genome

Genomes

Upload target gene

Fasta format. Sequence of expressed region is required for eukaryotic genes.

Upload inactivated target gene

Email address

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.