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Genome Editing


Cas9 expression/mRNA production plasmid

Catalog no. Name of clone Running title
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).
Depositor: Dr. Tomoji Mashimo

References and Related Articles:

  • Yoshimi, K., ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes. Nat. Commun. 7: 10431 (2016). PMID 26786405.
  • Miyasaka, Y., CLICK: one-step generation of conditional knockout mice. BMC Genomics 19 (1): 318 (2018). PMID 29720086.

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.


Delivatives of the T7-NLS hCas9-pA (RDB13130)

Catalog no. Name of clone Running title
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.

Depositor: DNA Bank, RIKEN BRC

 


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

Depositor: Dr. Yasunori Sasakura
References and Related Articles:

  • Sasaki, H., CRISPR/Cas9-mediated gene knockout in the ascidian Ciona intestinalis. Dev. Growth Differ. 56 (7): 499-510 (2014). PMID 25212715.

 

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.

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

Depositor: Dr. Fumihiro Sugiyama
References and Related Articles:

  • Hasegawa, Y., Generation of CRISPR/Cas9-mediated bicistronic knock-in ins1-cre driver mice. Exp. Anim. 65 (3): 319-327 (2016). PMID 27053096.

Catalog no. Name of clone Running title
RDB13532 pCAG-EGxxFP Validation plasmid for the effectiveness of guide RNA in CRISPR/Cas9 system.

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.
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.
Depositor: Dr. Masahito Ikawa
References and Related Articles:

  • Mashiko, D., Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Sci. Rep. 3: 3355 (2013). PMID 24284873.

Catalog no. Name of clone Running title
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.
Depositor: Dr. Yu Hisano
References and Related Articles:

  • Hisano, Y., Quantitative assay for TALEN activity at endogenous genomic loci. Biology Open., 2: 363-367 (2013). PMID 23616919.

 

Resources for CRISPR/Cas9 genome editing technology

Knock-in donors and tags utilized in the genome editing

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

Depositor: Dr. Masato Kinoshita

References and Related Articles:

  • Murakami, Y., An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes). Zoological Lett. 3: 10 (2017). PMID 28694996.

Catalog no. Name of clone Running title
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.

Depositor: Dr. Atsuo Kawahara
References and Related Articles:

  • Hisano, Y., Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish. Sci. Rep. 5: 8841 (2015). PMID 25740433.

Selection marker Attached tag at the C-terminus
mClover mCherry2 S tag-3FLAG
Neo pMK277 pMK280 pMK283
Hygro pMK278 pMK281 pMK284
Bsr pMK279 pMK282 pMK285

Please refer Fig. 4 of Natsume, T., Cell Reports 15, 210-218 (2016) for an instruction about how to use these clones.
Depositor: Dr. Masato Kanemaki
References and Related Articles:

  • Natsume, T., Rapid protein depletion in human cells by auxin-inducible degron tagging with short homology donors. Cell Rep. 15 (1): 210-218 (2016). PMID 27052166.

Please visit the Auxin Inducible Degron (AID) System.

(GRP0024e 2016.01.19 N.N.)

2018.10.18