TadA-8e and codon-optimized engineered orthologous TadAs were synthesized commercially (Synbio Technologies). The mutagenesis strategy (KOD-plus, Toyobo, Cat#: KOD-201) was used to induce specific mutations for corresponding base editors. To generate TadA ortholog-derived base editors, the docking site at residue 1249 was initially inserted with SpeI-NLS-BamHI-NheI-linker-XbaI sequence, and then engineered orthologous TadAs were inserted via BamHI-NheI double digestion strategy. Plasmids expressing dSaCas9-UGI-T2A-mCherry and U6-sgsaRNA, which were used in R-loop assay, were derived from PX602 (Addgene #61593), in which D10A and N580A were induced via mutagenesis strategy (KOD-plus, Toyobo, Cat#: KOD-201), and then UGI-T2A-mCherry cassette was inserted through BamHI-EcoRI double digestion strategy. SgRNA expression vectors were U6-sgRNA-EF1alpha-UGI-T2A-mCherry as described previously, and to generate plasmid expressing sgRNAs for puromycin-based enrichment, mCherry was replaced with a puromycin resistance gene. Information for sgRNAs used in this study was listed in Supplementary Data 4 .
Px602
Manufactured by Addgene
The PX602 is a high-performance lab equipment designed for a variety of scientific applications. It provides precise control and measurement capabilities for conducting experiments and analyses. The core function of the PX602 is to facilitate accurate data collection and monitoring within laboratory settings.
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Lab products found in correlation
3 protocols using px602
1
Engineered Orthologous TadAs-Derived Base Editors
2
AAV-mediated CasRx delivery for Gm19619 knockdown
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The plasmid of pAAV-EFS-CasRx was constructed by cloning the EFS promoter and RfxCas13d from the pLentiRNACRISPR_005 plasmid (Addgene #138147) and replaced the TBG promoter and SaCas9 gene in px602 plasmid (Addgene #61593). The DR30 sgRNA cassette for CasRx sgRNA cloning was used to further replace the Sa gRNA scaffold. Five individual sgRNA candidates targeting Gm19619 was inserted after the DR30 respectively and verified their knockdown efficacy in 293 T cells against the co-transfected pCMV-Gm19619 plasmid by QPCR. Then three sgRNAs were chosen to be concatenated and spaced by DR36 sequences to construct the pAAV-EFS-CasRx-19×3 plasmid for AAV packaging. Four-week-old C57BL/6 mice were fed with HFD for 3 weeks, then received once intravenous injections of 5 × 1011 genome copies of AAV-CasRx-19×3, as well as AAV-CasRx-GFP for control and continued on HFD until euthanization.
3
Engineered SaCas9 and AAV Constructs
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Annealed guide sequence oligos were inserted to the Bsa1 digested site of pX602 (Addgene plasmid #61593) to construct SaCas9-sgRNA1, sgRNA2, and sgRNA3, respectively. GFP-IRES-PuroR was cloned into the AAV vector (Addgene plasmid #60229) to replace the Cre site for constructing AAV-CBh-GFP, with the same done for AAV-CBh-mKate2. The TBG promoter of pX602 was replaced by human synapsin I promoter. The capsid vector ShH10 was a gift from David V. Schaffer (UC Berkeley). Individual AAVs were produced by transfecting HEK293T cells with three plasmids, i.e., AAV vector, ShH10, and pHelper. The viruses in the nucleus were harvested and purified by iodixanol gradient ultracentrifugation (32 000 rpm, 4 h, 18 °C) in a SW32 rotor, and then concentrated with an Amicon Ultra-15 filter (Millipore). The final titers of AAVs were 5-12 × 10 12 virus genome/ml, as quantified by qPCR, and all viruses were adjusted to the same titer (5× 10 12 virus genome/ml) before injection.
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