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CRISPR

VectorBuilderを使って自分のベクターをデザインすることができます。以下によく使われるコンポーネントの解説があります。
Cas Proteins
名前 詳細 アプリケーションノート PAM Sequence レファレンス シークエンス
zCas9 Zebrafish codon-optimized version of S. pyogenes Cas9 with SV40 large T-antigen nuclear localization signals (nls) at both its amino and carboxyl termini Generates double-strand DNA breaks. NGG (preferred); NAG (less preferred) Proc Natl Acad Sci USA. 110: 13904 (2013) View
hCas9 Human codon-optimized CRISPR associated protein 9 from Streptococcus pyogenes (with codon-optimized 3xFLAG tag and nuclear localization signal), also known as SpCas9 Generates double-strand DNA breaks. NGG (preferred); NAG (less preferred) Science. 339:819 (2013) View
Cas9(D10A) Human codon-optimized CRISPR associated protein 9 with D10A mutation, HA tag and nuclear localization signal Nickase; generates 5' overhang; can induce NHEJ with paired offset gRNAs; reduced off-target effect. NGG (preferred); NAG (less preferred) Cell. 154: 1380 (2013) View
dCas9 Catalytically inactive Cas9, a variant of Cas9 bearing both D10A and H840A mutations Catalytically inactive but can be recruited by gRNAs to target sites; can be fused to other proteins to direct them to specific genomic locations (e.g. dCas9 fused with transcriptional activators or repressors can lead to activation or repression of target genes). NGG (preferred); NAG (less preferred) Nat Biotechnol. 32: 347 (2014) View
dCas9/VP64 dCas9 fused with a VP64 acidic transactivation domain Used for RNA-guided transcriptional activation NGG (preferred); NAG (less preferred) Proc Natl Acad Sci U S A. 97:1495 (2000); Nat Methods. 10:973 (2013) View
dCas9/VPR dCas9 fused with a tripartite VP64-p65-Rta (VPR) transactivation domain Used for RNA-guided transcriptional activation. NGG (preferred); NAG (less preferred) Nat Methods. 12:326 (2015) View
dCas9/KRAB dCas9 fused with a KRAB transcriptional silencing domain Used for RNA-guided transcriptional repression NGG (preferred); NAG (less preferred) Proc Natl Acad Sci U S A. 97:1495 (2000); Cell. 159:647 (2014) View
dCas9/KRAB/MeCP2 dCas9 fused with a carboxy terminal bipartite repressor domain, KRAB-MeCP2 Efficiently suppresses gene transcription by physically blocking RNA polymerase passage by dCas9 and further transcriptional inhibition by the KRAB-MeCP2 repressor domain. NGG (preferred); NAG (less preferred) Cell. 88:471 (1997); Nat Methods. 15:611 (2018) View
dCas9-10xGCN4_v4 dCas9 with SV40 large T-antigen nuclear localization signals (nls) at both its amino and carboxyl termini, fused to an optimized GCN4 antibody-peptide pair (containing v4 version of the GCN4 peptide array with 10 copies of the peptide binding site, also know as SunTag10x_v4) Modulates transcription of endogenous genes by recruitment of multiple copies of regulatory effector domains to genomic sites of interest targeted by dCas9 protein via binding of scFv antibody to a GCN4 repeating peptide array termed SunTag. NGG (preferred); NAG (less preferred) Cell. 159: 635 (2014) View
SpCas9-HF1 High-fidelity variant of SpCas9 Designed to reduce non-specific DNA contacts; fewer off-target cuts. NGG (preferred); NAG (less preferred) Nature. 529:490 (2016) View
eSpCas9 Enhanced specificity SpCas9 variant Low off-target effects and robust on-target cleavage. NGG (preferred); NAG (less preferred) Science. 351:84 (2016) View
BE3 rAPOBEC–XTEN–Cas9_D10A–UGI (rat cytidine deaminase enzyme fused with Cas9_D10A and uracil DNA glycosylase inhibitor via an XTEN linker) Converts cytosine (C) to uracil (U) between the 4th and 8th bases of the sgRNA binding site, targeting the nonbinding strand. The modified base can be permanently converted to thymine (T) following DNA replication or repair. NGG (preferred); NAG (less preferred) Nature. 533:420 (2016); Nat Methods. 14:710 (2017) View
Cas12a Also known as Cpf1; single RNA-guided endonuclease of a class 2 CRISPR-Cas system from Acidaminococcus, human codon-optimized, and linked to an HA tag and nuclear localization signal Cleaves DNA via a staggered DNA double strand break. Able to process its own CRISPR RNA (crRNA), which can simplify multiplexed genome editing. TTTN Cell. 163:759 (2015);Nat Biotechnol. 35:31 (2017) View
Cas9/mSA hCas9 fused with monomeric streptavidin Used for efficient generation of targeted large insertions using biotinylated repair templates. NGG (preferred); NAG (less preferred) Biotechnol Bioeng. 110:57 (2013); Nat Biotechnol. 36:632 (2018) View
SaCas9 Staphylococcus aureus CRISPR associated protein 9 with HA tag and nuclear localization signal Shorter sequence; especially useful in vector systems that have limited cargo space (e.g. AAV vectors). NNGRR; NNGRRT(preferred) Nature. 520:186 (2015) View
hCas9:T2A:Neo hCas9 and neomycin resistance gene linked by T2A Allows cells to express hCas9 protein and be resistant to neomycin. NGG (preferred); NAG (less preferred) Engineered by VectorBuilder View
hCas9:T2A:Puro hCas9 and puromycin resistance gene linked by T2A Allows cells to express hCas9 protein and be resistant to puromycin. NGG (preferred); NAG (less preferred) Engineered by VectorBuilder View
hCas9:T2A:Hygro hCas9 and hygromycin resistance gene linked by T2A Allows cells to express hCas9 protein and be resistant to hygromycin B. NGG (preferred); NAG (less preferred) Engineered by VectorBuilder View
hCas9:T2A:Bsd hCas9 and blasticidin resistance gene linked by T2A Allows cells to express hCas9 protein and be resistant to blasticidin. NGG (preferred); NAG (less preferred) Engineered by VectorBuilder View
hCas9:P2A:EGFP hCas9 and EGFP linked by P2A Allows cells to express hCas9 protein and be visualized by green fluorescence. NGG (preferred); NAG (less preferred) Engineered by VectorBuilder View
hCas9:P2A:mCherry hCas9 and mCherry linked by P2A Allows cells to express hCas9 protein and be visualized by red fluorescence. NGG (preferred); NAG (less preferred) Engineered by VectorBuilder View
DmCas9 Fruit fly (Drosophila melanogaster) codon-optimized CRISPR associated protein 9 with nuclear localization signal Generates double-strand DNA breaks. NGG (preferred); NAG (less preferred) Proc Natl Acad Sci U S A. 111:E2967 (2014) View
CjCas9 A small Cas9 orthologue derived from Campylobacter jejuni with a carboxy terminal nuclear localization signal and HA tag Shorter sequence; especially useful in vector systems that have limited cargo space (e.g. AAV vectors). NNNNR(A/G)Y(C/T)AC; NNNNACAC (most preferred) Nat Commun. 8:14500 (2017) View
CeCas9 S. pyogenes Cas9 codon-optimized for C. elegans, containing multiple synthetic introns, a carboxy terminal nuclear localization signal, and an HA tag Generates double-strand DNA breaks. NGG (preferred); NAG (less preferred) Nat Methods. 10:1028 (2013) View
Guide RNAs
名前 ガイドシークエンス ターゲット遺伝子 ターゲットポジション レファレンス シークエンス
Scramble[gRNA#1] GTGTAGTTCGACCATTCGTG None in human and mouse Engineered by VectorBuilder View
Scramble[gRNA#2] GTTCAGGATCACGTTACCGC None in human and mouse Engineered by VectorBuilder View
Scramble[SagRNA#1] GTGTAGTTCGACCATTCGTG None in human and mouse Engineered by VectorBuilder View
Scramble[SagRNA#2] GTTCAGGATCACGTTACCGC None in human and mouse Engineered by VectorBuilder View
Scramble[msgRNA#1] GTGTAGTTCGACCATTCGTG None in human and mouse Engineered by VectorBuilder View
Scramble[msgRNA#2] GTTCAGGATCACGTTACCGC None in human and mouse Engineered by VectorBuilder View
EGFP[gRNA#1] GGGCGAGGAGCTGTTCACCG EGFP 12-31 Science. 343:84 (2014) View
EGFP[gRNA#2] GAAGTTCGAGGGCGACACCC EGFP 339-358 Science. 343:84 (2014) View
ROSA26[gRNA#1] CGCCCATCTTCTAGAAAGAC Mouse ROSA26 locus 1st intron Designed by VectorBuilder View
AAVS1[gRNA#1] GGGGCCACTAGGGACAGGAT Human AAVS1 locus 1st intron Science. 339:823 (2013) View
EMX1[SagRNA#1] GGCCTCCCCAAAGCCTGGCCA Human EMX1 3rd exon Engineered by VectorBuilder View
Scramble[msSagRNA#1] GTGTAGTTCGACCATTCGTG None in human and mouse Engineered by VectorBuilder View
Scramble[msSagRNA#2] GTTCAGGATCACGTTACCGC None in human and mouse Engineered by VectorBuilder View
ttTi5605[gRNA#1] GATATCAGTCTGTTTCGTAA C. elegans ttTi5605 locus Adjacent to the ttTi5605 Mos1 insertion site on chromosome II Nat Methods. 10:1028 (2013) View
CRISPR Target Sequences
名前 Target Sequence + PAM ターゲット遺伝子 ターゲットポジション レファレンス シークエンス
Scramble[gRNA#1]_target GTGTAGTTCGACCATTCGTGTGG None in human and mouse Engineered by VectorBuilder View
ROSA26[gRNA#1]_target CGCCCATCTTCTAGAAAGACTGG Mouse ROSA26 locus 1st intron Designed by VectorBuilder View
AAVS1[gRNA#1]_target GGGGCCACTAGGGACAGGATTGG Human AAVS1 locus 1st intron Science. 339:823 (2013) View
Scramble[SagRNA#1]_target GTGTAGTTCGACCATTCGTGGGGAGT None in human and mouse Engineered by VectorBuilder View
EMX1[SagRNA#1]_target GGCCTCCCCAAAGCCTGGCCAGGGAGT Human EMX1 3rd exon Nature. 520:186 (2015) View
Homology Arms
名前 詳細 アプリケーションノート レファレンス シークエンス
mROSA26_LA Left homology arm targeting mouse ROSA26 Pairs with mROSA26_RA to target the safe harbor site at intron 1 of mouse ROSA26 locus. Methods Mol Biol.667:145 (2010) View
mROSA26_RA Right homology arm targeting mouse ROSA26 Pairs with mROSA26_LA to target the safe harbor site at intron 1 of mouse ROSA26 locus. Methods Mol Biol.667:145 (2010) View
hAAVS1_LA Left homology arm targeting human AAVS1 Pairs with hAAVS1_RA to target the safe harbor site at human AAVS1 locus. Nat Biotechnol.27:851 (2009) View
hAAVS1_RA Right homology arm targeting human AAVS1 Pairs with hAAVS1_LA to target the safe harbor site at human AAVS1 locus. Nat Biotechnol.27:851 (2009) View
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