...the second design (in pX330 / PX335 ), so we recommend people all use the pX330/PX335 system ....the oligo when cloning my target spacers into the PX330 vector backbone? There is no need to add the NGG...having a targeting spacer cloned into the backbone ( pX330 or PX335 , depending if you want to use wildtype...wildtype Cas9, clone two spacers for each locus into PX330, then co-transfect or delivery the two plasmids ...not a very big concern, you could go ahead with PX330 plasmid, clone in your target guides, test run them...into the cells with the Cas9/sgRNAs vector (like PX330). For large changes (>100bp insertions or deletions...if the cloning of my oligo into a backbone like PX330 worked? You can quickly check which of your colonies...

...we discuss the usage of the pSpCas9(BB) plasmid (pX330) (Addgene plasmid ID 42230). This plasmid allows...guide’s reverse complement for cloning into the pX330 vector using BbsI restriction enzyme ( Table 3 )...G. sgRNA expression from the U6 promoter of the pX330 vector is enhanced by the inclusion of a G nucleotide...concentration of 1 μM). Ligate annealed oligos into pX330 using a Golden Gate assembly cloning strategy 3 .... Prepare a 50 μl reaction mix: 100 ng circular pX330 vector, 1.0 μl annealed oligos (1 μM; see step 3.1.4...

...Yamamoto Tagging Plasmids: pX330S-2-PITCh - expresses Cas9 & PITCh-gRNA pX330A-FBL/PITCh - expresses Cas9...all-in-one CRISPR/Cas9 vector based on the Zhang lab's PX330 , simultaneously targeting the genomic site and ...gene-specific CRISPR and donor vectors as well as pX330S-2-PITCh, expressing a generic gRNA targeting the...

... Deletions in Mammalian Cell Lines pSpCas9(BB) (pX330) Link Parrott NEBuilder Assembly of CRISPR vectors... (pX260) or Cas9 D10A (pX334) ; tracrRNA: Cas9 (pX330) or Cas9 D10A (pX335) ; PDF 248.6 KB Zhang sgRNA...

...Mammalian BbsI yes, nick S. pyogenes Puro Zhang PX330 (2nd Gen) 42230 Mammalian BbsI yes, cut S. pyogenes...Mammalian U6 yes, methylation S. pyogenes Puro Zoldos pX330-U6-Chimeric_BB-CBh-hSpCas9-hGem(1/110) 71707 Mammalian...

...plasmids can be ordered via the links below: 42230 : PX330; SpCas9 and single guide RNA 48138 : PX458; SpCas9...

...G2019S-ng LRRK2 U6 Parkinson's Frank Soldner 180437 Px330-EGFP-LRRK2-CRISPR/Cas9 LRRK2 U6 Parkinson's Frank...