Identification of Cas9 homologs and generation of plasmids
This protocol is extracted from research article:
Minimal PAM specificity of a highly similar SpCas9 ortholog
Sci Adv, Oct 24, 2018; DOI: 10.1126/sciadv.aau0766

The UniProt database (16) was mined for all Streptococcus Cas9 protein sequences, which were used as inputs to either the BioPython pairwise2 module or Geneious to conduct global pairwise alignments with SpCas9, using the BLOSUM62 scoring matrix (17) and subsequently calculate percent homology. The ScCas9 was codon optimized for Escherichia coli, ordered as multiple gBlocks from Integrated DNA Technologies and assembled using Golden Gate Assembly. The pSF-EF1-Alpha-Cas9WT-EMCV-Puro (OG3569) plasmid for human expression of SpCas9 was purchased from Oxford Genetics, and the ORFs of Cas9 variants were individually amplified by polymerase chain reaction (PCR) to generate 35–base pair (bp) extensions for subsequent Gibson Assembly into the OG3569 backbone. Engineering of the coding sequence of ScCas9 and SpCas9 for removal or insertion of motifs was conducted using either the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) or Gibson Assembly. To generate ScCas9 base-editing plasmids, pCMV-ABE(7.10) (Addgene plasmid no. 102919) and pCMV-BE3 (Addgene plasmid no. 73021) were received as gifts from D. Liu. Similarly, the ORF of the ScCas9 D10A nickase was amplified by PCR to generate 35-bp extensions for subsequent Gibson Assembly into each base-editing architecture backbone. sgRNA plasmids were constructed by annealing oligonucleotides coding for crRNA sequences (table S1) and 4-bp overhangs and subsequently performing a T4 DNA ligase–mediated ligation reaction into a plasmid backbone immediately downstream of the human U6 promoter sequence. Assembled constructs were transformed into 50 μl of NEB Turbo Competent E. coli cells and plated onto Luria broth (LB) agar supplemented with the appropriate antibiotic for subsequent sequence verification of colonies and plasmid purification.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.