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 | |
---|---|---|---|
Cow | Bos taurus | Ensembl 108 | ARS-UCD1.2 |
Worm | Caenorhabditis elegans | Ensembl 108 | WBcel235 |
Marmoset | Callithrix jacchus | Ensembl 108 | mCalJac1.pat.X |
Ciona | Ciona intestinalis | Ensembl 108 | KH |
Zebrafish | Danio rerio | Ensembl 108 | GRCz11 |
Zebrafish NHGRI (SNP only) | Danio rerio nhgri snp | Nhgri | GRCz11 |
Fly | Drosophila melanogaster | Ensembl 108 | BDGP6.32 |
Chicken | Gallus gallus | Ensembl 108 | bGalGal1.mat.broiler.GRCg7b |
Human | Homo sapiens | Ensembl 108 | GRCh38 |
Green sea urchin | Lytechinus variegatus | Ensembl genomes 55 | Lvar_3.0 |
Sea walnut | Mnemiopsis leidyi | Ensembl genomes 55 | MneLei_Aug2011 |
Mouse | Mus musculus | Ensembl 108 | GRCm39 |
Sea anemone | Nematostella vectensis | Ensembl genomes 55 | ASM20922v1 |
Killifish | Nothobranchius furzeri | Brunet | NotFur1 |
Medaka | Oryzias latipes | Ensembl 108 | ASM223467v1 |
Chimpanzee | Pan troglodytes | Ensembl 108 | Pan_tro_3.0 |
Bat star | Patiria miniata | Ensembl genomes 55 | GCA015706575v1 |
Rat | Rattus norvegicus | Ensembl 108 | mRatBN7.2 |
Yeast | Saccharomyces cerevisiae | Ensembl 108 | R64-1-1 |
Purple sea urchin | Strongylocentrotus purpuratus | Ensembl genomes 55 | Spur_5.0 |
Frog | Xenopus laevis | Xenbase | Xenopus_laevis_v10.1 |
Frog | Xenopus tropicalis | Ensembl 108 | UCB_Xtro_10.0 |
Plasmid for targeting Cas9 expression into the germ line is available at Addgene.