CRISPRscan

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.

Off-target predictions

CRISPRscan searches for potential genomic off-targets with the following rules.

Cas9

All: According to Hsu et al. Nature Biotechnology 2013, potential off-targets can have a maximum of 2 mismatches with the sgRNA.
Seed: With the method published by Cong et al. Science 2013, potential off-targets must match perfectly in their seed (12 nt 3' of the PAM sequence) and a maximum of 2 mismatches in the rest of the sgRNA. This rule is more stringent than the All method and therefore less off-targets are found.
CFD (Cutting Frequency Determination): Doench et al. Nature Biotechnology 2016 measured the cutting efficiency of potential off-targets and integrated them into the CFD score. Potential off-targets with up to 4 mismatches are scored with Doench et al. matrix.

Cpf1

All: Potential off-targets can have a maximum of 2 mismatches with the gRNA.
Seed: The experiments published by Kim et al. Nature Methods 2017 support potential off-targets that must match perfectly in their seed (6 nt 3' of the PAM sequence) and a maximum of 2 mismatches in the rest of the gRNA. This rule is more stringent than the All method and therefore less off-targets are found.

gRNA generation

Detailed information can be found in our protocols:

For Cas9: Optimized CRISPR–Cas9 system for genome editing in zebrafish
For Cas12a: Optimized CRISPR-Cpf1 system for genome editing in zebrafish

Cas9

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

Cpf1

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
Oligo containing the target sequence are reported 5' to 3' ready to be ordered in the "Oligo" column, i.e. Cpf1 crRNA primer is reported reverse-complement of the target as shown above. CCC upstream of promoter are optional aiming to increase stability of oligo.

Genomes and genes

Sequences of genomes and annotations of genes were obtained from these sources:

Name		Source	Assembly
Chicken	Gallus gallus	Ensembl 102	GRCg6a
Chimpanzee	Pan troglodytes	Ensembl 102	Pan_tro_3.0
Ciona	Ciona intestinalis	Ensembl 102	KH
Cow	Bos taurus	Ensembl 102	ARS-UCD1.2
Fly	Drosophila melanogaster	Ensembl 102	BDGP6.28
Frog	Xenopus laevis	Xenbase	Xenopus_laevis_v2
Frog	Xenopus tropicalis	Xenbase	Xenopus_tropicalis_v10.0
Human	Homo sapiens	Ensembl 102	GRCh38
Killifish	Nothobranchius furzeri	Brunet	NotFur1
Marmoset	Callithrix jacchus	Ensembl 102	ASM275486v1
Medaka	Oryzias latipes	Ensembl 102	ASM223467v1
Mouse	Mus musculus	Ensembl 102	GRCm38
Rat	Rattus norvegicus	Ensembl 102	Rnor_6.0
Sea anemone	Nematostella vectensis	Ensembl genomes 49	ASM20922v1
Sea urchin	Strongylocentrotus purpuratus	Ensembl genomes 49	Spur_5.0
Sea walnut	Mnemiopsis leidyi	Ensembl genomes 49	MneLei_Aug2011
Worm	Caenorhabditis elegans	Ensembl 102	WBcel235
Yeast	Saccharomyces cerevisiae	Ensembl 102	R64-1-1
Zebrafish	Danio rerio	Ensembl 102	GRCz11

Plasmid: Cas9 with nanos 3’-UTR

Plasmid for targeting Cas9 expression into the germ line is available at Addgene.