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 | ||
|
Sequences of genomes and annotations of genes were obtained from these sources:
Name | Source | Assembly | |
---|---|---|---|
Zebrafish | Danio rerio | Ensembl 104 | GRCz11 |
Zebrafish NHGRI (SNP only) | Danio rerio nhgri snp | Nhgri | GRCz11 |
Fly | Drosophila melanogaster | Ensembl 104 | BDGP6.32 |
Worm | Caenorhabditis elegans | Ensembl 104 | WBcel235 |
Human | Homo sapiens | Ensembl 104 | GRCh38 |
Medaka | Oryzias latipes | Ensembl 104 | ASM223467v1 |
Yeast | Saccharomyces cerevisiae | Ensembl 104 | R64-1-1 |
Sea walnut | Mnemiopsis leidyi | Ensembl genomes 51 | MneLei_Aug2011 |
Mouse | Mus musculus | Ensembl 104 | GRCm39 |
Sea anemone | Nematostella vectensis | Ensembl genomes 51 | ASM20922v1 |
Rat | Rattus norvegicus | Ensembl 104 | Rnor_6.0 |
Sea urchin | Strongylocentrotus purpuratus | Ensembl genomes 51 | Spur_5.0 |
Frog | Xenopus laevis | Xenbase | Xenopus_laevis_v2 |
Frog | Xenopus tropicalis | Xenbase | Xenopus_tropicalis_v10.0 |
Killifish | Nothobranchius furzeri | Brunet | NotFur1 |
Cow | Bos taurus | Ensembl 104 | ARS-UCD1.2 |
Marmoset | Callithrix jacchus | Ensembl 104 | ASM275486v1 |
Ciona | Ciona intestinalis | Ensembl 104 | KH |
Chicken | Gallus gallus | Ensembl 104 | GRCg6a |
Chimpanzee | Pan troglodytes | Ensembl 104 | Pan_tro_3.0 |
Plasmid for targeting Cas9 expression into the germ line is available at Addgene.