Synthesis of Cas9 mRNA and sgRNAs

For synthesis of Cas9 mRNA in vitro, plasmid vector pCAG-T3-hCAS-pA (Addgene 48625) was linearized by Sph I, then transcribed with T3 RNA polymerase (Promega) in the presence of Ribo m⁷G Cap Analog (promega) as previously described [31 (link)]. The MEGAshortscript T7 (Thermo Fisher Scientific AM1354) and MEGAclear (Thermo Fisher Scientific AM1908) kits were used for in vitro transcription of sgRNAs, while the CRISPR Design tool was used for creating sgRNAs [32 (link)]. All oligonucleotide sequences used for in vitro transcription are listed in Supplementary Table S4.

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Watamura N., Kakiya N., Nilsson P., Tsubuki S., Kamano N., Takahashi M., Hashimoto S., Sasaguri H., Saito T, & Saido T.C. (2021). Somatostatin-evoked Aβ catabolism in the brain: Mechanistic involvement of α-endosulfine-KATP channel pathway. Molecular Psychiatry, 27(3), 1816-1828.

Publication 2021

pCAG-T3-hCAS-pA T3 RNA polymerase Ribo m7G Cap Analog MEGAshortscript T7 MEGAclear

Crispr Mrna Oligonucleotide Plasmid Promega Synthesis T3 rna polymerase Transcription Vector

Corresponding Organization : RIKEN Center for Brain Science

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Variable analysis

independent variables

Linearization of plasmid vector pCAG-T3-hCAS-pA with Sph I
Transcription of Cas9 mRNA with T3 RNA polymerase in the presence of Ribo m⁷G Cap Analog
In vitro transcription of sgRNAs using the MEGAshortscript T7 and MEGAclear kits
Creation of sgRNAs using the CRISPR Design tool

dependent variables

Synthesis of Cas9 mRNA in vitro
Synthesis of sgRNAs in vitro

control variables

Oligonucleotide sequences used for in vitro transcription (listed in Supplementary Table S4)

controls

No positive or negative controls were explicitly mentioned in the provided information.

Annotations

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