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eci2 knockout in Zebrafish using CRISPR-Cas9 System

131028 Mie Meeting Research, Yasuhito Shimada. eci2 knockout in Zebrafish using CRISPR-Cas9 System . The diverse potential application of CRISPR-Cas9 system. Nat Methods. 10: 957-963, 2013 (Figure 2C). Purpose: To construct eci2 knockout zebrafish. The CRISPR Design Tools

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eci2 knockout in Zebrafish using CRISPR-Cas9 System

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  1. 131028 Mie Meeting Research, Yasuhito Shimada eci2 knockout in Zebrafish using CRISPR-Cas9 System The diverse potential application of CRISPR-Cas9 system. Nat Methods. 10: 957-963, 2013 (Figure 2C) Purpose: To construct eci2 knockout zebrafish. The CRISPR Design Tools ZiFitTargeter Version 4.2 (ZINC Finger Consortium, Joung Lab, Massachusetts General Hospital): Nat Biotechnol. 31: 227-229, 2013 CRISPR Design (Zhang Lab, Broad Institute of MIT and Harvard): Nat Biotechnol. 31: 827-832, 2013 ① ② ③ eci2 mRNA sequence (not genome sequence)

  2. ④ Results Check by Genome-blast Remove the oligo covered to exon-intron boundary ⑤ Positive controls fh: Zebrafish-gRNA-0004 (site #2) gsk3b: Zebrafish-gRNA-0005 (Nat Biotechnol. 2013, Supplementary) Plasmid Purchase from Addgene Cut: Cas9 nucleases Nick: Cas9 nickases Interfere: CRISPR interference (CRISPRi) Active: dCas9-activator fusion

  3. ⑥ Amp Kan Plasmid Construction DR274 linearization using BsaI ⑦ ⑧ 2.1kbp 37℃, 1h 65℃, 20min Electrophoresis on 1.5% Agarose gel Gel Extraction using Wizard SV Gel and PCR Clean-Up System (Promega) Measure the concentration using NanoDrop (35ng/μl in 50 μl) Annealing for insert oligonucleotides using 1 ×annealing solution (Life Technologies) ⑨ Ligation using Ligation-convenience Kit (Wako Pure chemicals) and Transformation Plasmid : Insert DNA = 1 : 5 The size of plasmid = 2147 - extra bp = 2100 bp The MW = 660 x 2100 = 1386000 34.37 ng/ul = 24.8 nM Transformation DR274 (positive control) DR274-linealized Ligated DR274 (gsk3b) 10 min at 16℃.

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