CRISPRscan is a novel algorithm to predict gRNA efficiency.
Based on a large scale analysis of sgRNA mutagenesis activity in zebrafish, we established rules to predict sgRNA activity in vivo and build the CRISPRscan model integrating these rules. We independently validated with success our predictions using sgRNAs different from the large scale analysis.
CRISPRscan searches for potential genomic off-targets with the following rules.
Detailed information can be found in our protocols:
Oligo sequences | |||
---|---|---|---|
Oligo 1. sgRNA primer | T7 promoter | Target sequence | Tail annealing sequence |
5’ TAATACGACTCACTATA | GGNNNNNNNNNNNNNNNNNN | GTTTTAGAGCTAGAA | |
alternative promoter | Sp6 promoter | ||
5’ ATTTAGGTGACACTATA | GANNNNNNNNNNNNNNNNNN | GTTTTAGAGCTAGAA | |
Oligo 2. Tail primer | Tail | Tail annealing sequence | |
5’ AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTAT | TTCTAGCTCTAAAAC |
Oligo sequences | ||
---|---|---|
LbCpf1 | ||
Oligo 1. Tail primer | T7 promoter | Tail sequence |
5’ CCCTAATACGACTCACTATA | GGTAATTTCTACTAAGTGTAGAT | |
Oligo 2. crRNA primer | Target sequence (reverse) | Tail sequence |
5’ NNNNNNNNNNNNNNNNNNNNNNN | ATCTACACTTAGTAGAAATTA | |
AsCpf1 | ||
Oligo 1. Tail primer | T7 promoter | Tail sequence |
5’ CCCTAATACGACTCACTATA | GGTAATTTCTACTCTTGTAGAT | |
Oligo 2. crRNA primer | Target sequence (reverse) | Tail sequence |
5’ NNNNNNNNNNNNNNNNNNNNNNN | ATCTACAAGAGTAGAAATTA | |
NB | ||
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Sequences of genomes and annotations of genes were obtained from these sources:
Name | Source | Assembly | |
---|---|---|---|
Arabidopsis thaliana | Arabidopsis thaliana | Ensembl genomes 57 | TAIR10 |
Cow | Bos taurus | Ensembl 110 | ARS-UCD1.2 |
Worm | Caenorhabditis elegans | Ensembl 110 | WBcel235 |
Marmoset | Callithrix jacchus | Ensembl 110 | mCalJac1.pat.X |
Ciona | Ciona intestinalis | Ensembl 110 | KH |
Zebrafish | Danio rerio | Ensembl 110 | GRCz11 |
Zebrafish NHGRI (SNP only) | Danio rerio | Nhgri | GRCz11 |
Fly | Drosophila melanogaster | Ensembl 110 | BDGP6.46 |
Chicken | Gallus gallus | Ensembl 110 | bGalGal1.mat.broiler.GRCg7b |
Soybean | Glycine max | Ensembl genomes 57 | Glycine_max_v2.1 |
Human | Homo sapiens | Ensembl 110 | GRCh38 |
Green sea urchin | Lytechinus variegatus | Ensembl genomes 57 | Lvar_3.0 |
Sea walnut | Mnemiopsis leidyi | Ensembl genomes 57 | MneLei_Aug2011 |
Mouse | Mus musculus | Ensembl 110 | GRCm39 |
Sea anemone | Nematostella vectensis | Ensembl genomes 57 | ASM20922v1 |
Killifish | Nothobranchius furzeri | Brunet | NotFur1 |
Rice | Oryza sativa | Ensembl genomes 57 | IRGSP-1.0 |
Medaka | Oryzias latipes | Ensembl 110 | ASM223467v1 |
Chimpanzee | Pan troglodytes | Ensembl 110 | Pan_tro_3.0 |
Bat star | Patiria miniata | Ensembl genomes 57 | GCA015706575v1 |
Sea lamprey | Petromyzon marinus | Stowers | kPetmar1 |
Rat | Rattus norvegicus | Ensembl 110 | mRatBN7.2 |
Yeast | Saccharomyces cerevisiae | Ensembl 110 | R64-1-1 |
Sorghum | Sorghum bicolor | Ensembl genomes 57 | Sorghum_bicolor_NCBIv3 |
Purple sea urchin | Strongylocentrotus purpuratus | Ensembl genomes 57 | Spur_5.0 |
Frog | Xenopus laevis | Xenbase | Xenopus_laevis_v10.1 |
Frog | Xenopus tropicalis | Ensembl 110 | UCB_Xtro_10.0 |
Corn | Zea mays | Ensembl genomes 57 | Zm-B73-REFERENCE-NAM-5.0 |
Plasmid for targeting Cas9 expression into the germ line is available at Addgene.