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Px330s 2 pitch

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The PX330S-2-PITCh is a plasmid designed for CRISPR-Cas9 genome editing. It contains the SpCas9 gene and two guide RNA expression cassettes.

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Multiplex crispr cas9 assembly system kit, by Addgene (1 mentions)

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PX330 (156 citations)

2 protocols using px330s 2 pitch

1

CRISPR-Cas9 Knock-in Vector Construction

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pX330A-AAVS1/PITCh, an all-in-one CRISPR-Cas9 vector for cutting the genomic AAVS1 locus and the donor vector, was constructed using the Multiplex CRISPR/Cas9 Assembly System Kit (Addgene, Kit #1000000055) and pX330S-2-PITCh (Addgene, Plasmid #63670), in accordance with a described previously protocol.2 (link) The oligonucleotides for the template of sgRNA targeting AAVS1 are listed in Supplemental Table 1. pCRIS-PITChv2-AAVS1, the donor vector for PITCh-knock-in, was constructed using a standard PCR and In-Fusion cloning method (Takara).
Nakamae K., Nishimura Y., Takenaga M., Nakade S., Sakamoto N., Ide H., Sakuma T, & Yamamoto T. (2017). Establishment of expanded and streamlined pipeline of PITCh knock-in – a web-based design tool for MMEJ-mediated gene knock-in, PITCh designer, and the variations of PITCh, PITCh-TG and PITCh-KIKO. Bioengineered, 8(3), 302-308.
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2

Cloning PITCh Vectors for CRISPR-Mediated Genome Editing

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Cloning of PITCh vectors is based on ref. 52 (link). “pX330S-2-PITCh” (Addgene, plasmid no. 63670) containing PITCh gRNA was cloned via Golden Gate assembly into the “pX330A-1x2” vector (Addgene, plasmid no. 58766) expressing Cas9 and the gRNA against a target locus. Knock-in cassette flanked by 40 bp micro-homology arms were cloned into the “pCRIS-PITChv2-FBL” vector (Addgene, Plasmid no. 63672). 20 μg total (13 μg pX330A-1x2 : 7 μg pCRIS-PITChv2-FBL) were electroporated into 5x106 cells via the Maxcyte STX. Follow up steps were similarly performed as described in the previous section “Cloning and characterization of genome editing events”.
Neumayr C., Haberle V., Serebreni L., Karner K., Hendy O., Boija A., Henninger J.E., Li C.H., Stejskal K., Lin G., Bergauer K., Pagani M., Rath M., Mechtler K., Arnold C.D, & Stark A. (2022). Differential cofactor dependencies define distinct types of human enhancers. Nature, 606(7913), 406-413.
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