Genome Editing

RIKEN BRC has been permitted to collect and distribute bioresources developed using the CRISPR/Cas9 genome editing technology for academic research but not for commercial purpose by the Broad Institute of Harvard and MIT. We are looking forward to your deposition of CRISPR/Cas9 genome editing tools as well as request.

Cas9 expression/mRNA production plasmid

ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes.

Yoshimi, K., Kunihiro, Y., Kaneko, T., Nagahora, H., Voigt, B., Mashimo, T.
Nat. Commun. 7: 10431 (2016). PubMed PMID 26786405.

Catalog no. Name of clone Description
RDB13130 T7-NLS hCas9-pA Cas9-poly(A) expressing improved plasmid.

T7-NLS-hCAS9-pA is a plasmid for transcribing Cas9 mRNA with poly(A) tail allowing efficient Cas9 expression. Large-sized foreign genes were knock-in by introducing long single strand DNA together with the transcribed Cas9 mRNA into fertilized mouse and rat eggs by injection (Yoshimi, K. et al., 2016) and by electroporation (Miyasaka, Y. et al., 2018).

References and Related Articles:

  • Miyasaka, Y., CLICK: one-step generation of conditional knockout mice. BMC Genomics 19 (1): 318 (2018). PMID 29720086.

Delivatives of the T7-NLS hCas9-pA (RDB13130)
Catalog no. Name of clone Description
RDB14419 T7-NLS-hCAS9(D10A)-NLS-pA Cas9-poly(A) expressing improved plasmid.
RDB14420 T7-NLS-hCAS9(ver1.1)-NLS-pA Cas9-poly(A) expressing improved plasmid.
RDB14421 T7-NLS-hCAS9(D10A_ver1.1)-NLS-pA Cas9-poly(A) expressing improved plasmid.
RDB14657 T7-NLS-hCAS9-NLS-P2A-EGFP-pA Cas9-poly(A) expressing improved plasmid.
RDB14658 T7-NLS-hCAS9-NLS-P2A-mCherry-pA Cas9-poly(A) expressing improved plasmid.

A genome editing vector that enables easy selection and identification of knockout cells.

Nagasaki, A., Kato, Y., Meguro, K., Yamagishi, A., Nakamura, C., Uyeda, T.Q.P.
Plasmid 98: 37-44 (2018). PubMed PMID 30196057.

Catalog no. Name of clone Description
RDB16763 pGedit Plasmid vector for high efficiency genome editing oligo DNA containing target sequence.

 


Rapid protein depletion in human cells by auxin-inducible degron tagging with short homology donors.

Natsume, T., Kiyomitsu, T., Saga, Y., Kanemaki, M.T.
Cell Rep. 15 (1): 210-218 (2016). PubMed PMID 27052166.

Catalog no. Name of clone Description
RDB13917 AAVS1 T2 CRIPR in pX330 A CRISPR/Cas plasmid for making DNA dounle-strand break at the human AAVS1 locus.

Please visit the Auxin Inducible Degron (AID) System.


Disruption of MeCP2 attenuates circadian rhythm in CRISPR/Cas9-based Rett syndrome model mouse.

Tsuchiya, Y., Minami, Y., Umemura, Y., Watanabe, H., Ono, D., Nakamura, W., Takahashi, T., Honma, S., Kondoh, G., Matsuishi, T., Yagita, K.
Genes Cells 20 (12): 992-1005 (2015). PubMed PMID 26456390.

Catalog no. Name of clone Description
RDB14013 pgRNA_cloning_BbsI Expression vector backbone of sgRNA with U6 promoter.

CRISPR/Cas9-mediated gene knockout in the ascidian Ciona intestinalis.

Sasaki, H., Yoshida, K., Hozumi, A., Sasakura, Y.
Dev. Growth Differ.56 (7): 499-510 (2014). PubMed PMID 25212715.

Catalog no. Name of clone Description
RDB12994 pHTB Cas9 In vitro production of Cas9 mRNA.
RDB12999 pSPCiEF1>Cas9 Expression plasmid of Cas9.

 

Plasmids for the evaluation of the efficiency of genome editing

It is important to design efficient guide RNA to obtain the success on genome editing. To solve a problem there are two vectors to evaluate a function of guide RNA.

Generation of CRISPR/Cas9-mediated bicistronic knock-in ins1-cre driver mice.

Hasegawa, Y., Hoshino, Y., Ibrahim, A.E., Kato, K., Daitoku, Y., Tanimoto, Y., Ikeda, Y., Oishi, H., Takahashi, S., Yoshiki, A., Yagami, K., Iseki, H., Mizuno, S., Sugiyama, F.
Exp. Anim. 65 (3): 319-327 (2016). PubMed PMID 27053096.

Catalog no. Name of clone Description
RDB13948 p2color Genome editing tool for confirming the activity of CRISPR/Cas9 system.

Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA.

Mashiko, D., Fujihara, Y., Satouh, Y., Miyata, H., Isotani, A., Ikawa, M.
Sci. Rep. 3: 3355 (2013). PubMed PMID 24284873.

  • pCAG-EGxxFP developed and deposited by Dr. Masahito Ikawa and his colleagues at Osaka University (Mashiko, D. et al., 2013) allows you to evaluate the efficiency of genome editing by fluorescence intensity.
Catalog no. Name of clone Description
RDB13532 pCAG-EGxxFP Validation plasmid for the effectiveness of guide RNA in CRISPR/Cas9 system.
  • First, insert the PCR amplified target sequence of genome editing into the EGFP gene in the vector. Next, transfect the constructed vector together with Cas9 expression and guide RNA expression vectors in animal cells. If your guide RNA works well, you will observe fluorescence of GFP, which is caused by the double stranded break (DSB) of the target sequence and restoration of GFP sequence.

pCAG-NexxoR, the plasmid to enhance the productivity of aimed gene KO cell by CRISPR-Cas9 genome editing.

  • Newly available plasmid pCAG-NexxoR, the carrier of a pseudo-target of CRISPR-Cas9, indicate neomycin resistance after genome editing by homology directed repair. The drug resistance conferred by this plasmid is good indicator for target gene KO on genome, and thus enables to concentrate the aimed gene KO cells by the drug. This plasmid was developed by Dr. Mika Terumitsu, Dr. Kazuo Hashido and their colleagues in National center of Neurology and Psyciatry, Japan.
Catalog no. Name of clone Description
RDB16803 pCAG-NexxoR Selection plasmid for the positive clone of CRISPR/Cas9 system in the culture cells.
  • This plasmid contains separated parts of neomycin-resistant gene tethered by MCS under CAG promoter. This derives from pCAG-EGxxFP (RDB13532), which contains separated parts of EGFP gene tethered by MCS under CAG promoter.
  • USAGE:
    (1) Cloning of monitoring plasmid, which is CRISPR-Cas9 target genomic region inserted into pCAG-NexxoR.
    (2) Transfection cells with authentic tools of CRISPR-Cas9 genome editing along with monitoring plasmid.
    (3) Selection of cells actively undergone genome editing by culturing in medium containing G418.
    RESULT:
    About 98% of cells were undergone genome editing on aimed genomic target site. Notably, about 2% of them were homozygous mutants. (personal communication with the developer, Dr. Mika Terumitsu)
  • Experimental data using pCAG-NexxoR (Fig.1, kindly provided by the developer, Dr. Mika Terumitsu)
    To address if pCAG-NexxoR derivative inducing G418 resistant can accumulate mutation in other region of same target sequence after CRISPR-Cas9 genome editing, another genome editing monitoring tool, pCAG-EGxxFP was co-transfected. Treated cells with G418, almost all survived cells were notably GFP positive, compared to lots of GFP negative cells appeared in G418 untreated cells, or a consistent drug resistant plasmid pCAG-NeoR was used instead of pCAG-NexxoR derivative. Suggesting that pCAG-NexxoR carrying pseudo-target works to monitor real-target mutation in CRISPR-Cas9 genome editing experiments.
  • Fig.1: Two different monitoring plasmid for CRISPR-Cas9 genome editing works at once in a cell.
    The same “target sequence” was inserted into both pCAG-EGxxFP and pCAG-NexxoR to monitor cells actively undergone genome editing by GFP and G418 resistant, respectively. Both monitoring plasmids along with pX330 derivative carrying gRNA attacking the “target sequence” were transfected into C2C12 cells. Cells were cultured in the medium with G418 (left panel), or without G418 (mid panel). (right panel) As left panel except pCAG-NeoR, which constitutively express neomycin resistant gene, were used instead of pCAG-NexxoR derivative.

Quantitative assay for TALEN activity at endogenous genomic loci.

Hisano, Y., Ota, S., Arakawa, K., Muraki, M., Kono, N., Oshita, K., Sakuma, T., Tomita, M., Yamamoto, T., Okada, Y., Kawahara, A.
Biol. Open. 2 (4): 363-367 (2013). PubMed PMID 23616919.

Catalog no. Name of clone Description
RDB12261 pBR-lacZ alpha Plasmid clone for quantitative assay for gene editing

pBR-lacZ alpha developed and deposited by Dr. Yu Hisano and his colleagues at RIKEN Quantitative Biology Center (QBiC) (Hisano, Y. et al., 2013) allows you to evaluate the efficiency of genome editing by the appearance of the blue/white colonies of E. coli.
After the induction of genome editing, amplify the target region by PCR and insert it into the lacZ gene in this vector. In the case you amplify the target region in frame on lacZ gene and then carry out transformation with the constructed vector, if your guide RNA works well, you can observe many of white colonies on agar plate supplemented with X-gal, IPTG and ampicillin.
 

Resources for CRISPR/Cas9 genome editing technology

Knock-in donors and tags utilized in the genome editing

An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes).

Murakami, Y., Ansai, S., Yonemura, A., Kinoshita, M.
Zoological Lett. 3: 10 (2017). PubMed PMID 28694996.

Catalog no. Name of clone Description
RDB15409 pBaitD-gap43-linker-EGFP Donor plasmid for targeted insertion into the gap43 locus of Japanese medaka by the BaitD system.

Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish.

Hisano, Y., Sakuma, T., Nakade, S., Ohga, R., Ota, S., Okamoto, H., Yamamoto, T., Kawahara, A.
Sci. Rep. 5: 8841 (2015). PubMed PMID 25740433.

Catalog no. Name of clone Description
RDB13480 pCS2P-tyr-mCherry-donor Donor vector for integrating mCherry into zebrafish tyrosinase locus.
RDB13481 pCS2P-krtt1c19e-linker-eGFP-mut-donor Donor vector for integrating eGFP mutant into zebrafish krtt1c19e locus.

Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.

Natsume, T., Kiyomitsu, T., Saga, Y., Kanemaki, M.T.
Cell Rep. 15 (1): 210-218 (2016). PubMed PMID 27052166.


(GRP0024e 2016.01.19 N.N.)

2020.11.12



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