Plasmid maxi kit

To determine DfCas9 PAM sequence a randomized 7N plasmid library carried a protospacer sequence flanked by seven randomized nucleotides was used (Supplementary Table S1). To create the library the ssDNA oligo Library_F containing randomized nucleotides was double-stranded through single stage PCR with Library_R primer (Evrogen). This fragment was assembled with PUC19 fragment synthesized through PCR using primers PUC19_F and PUC19_R by NEBuilder HiFi DNA Assembly Cloning Kit (NEB, E5520). The mix was transformed to Escherichia coli DH5alpha strain and plated to media supplemented with 100 μg/ml ampicillin. The plates were incubated at 37°C. Eighteen hours after transformation more than 50 000 colonies were washed off the plates, and the plasmid library was extracted by Qiagen Plasmid Maxi kit (Qiagen 12162). The library plasmid map is presented in the Supplementary Table S1. Competent E. coli Star cells carrying pACYC184_DfCas9_locus or an empty pACYC184 vector were transformed with 7N PAM plasmid libraries and plated to 100 μg/ml ampicillin and 25 μg/ml chloramphenicol containing agar plates. After 16 h cells were harvested and DNA was extracted using Qiagen Plasmid Maxi kit (Qiagen 12162). PAM-coding sequences were PCR amplified using M13_f and M13_r primers and sequenced using Illumina platform with pair-end 150 cycles (75+75).

Randomized 7N plasmid libraries carried a protospacer sequence flanked by seven randomized nucleotides (Supplementary Table S1). To create the library the ssDNA oligo Library_f containing randomized nucleotides was double-stranded through single stage PCR with Library_r primer (Evrogen). This fragment was assembled with PUC19 fragment synthesized through PCR using primers PUC19_F and PUC19_R by NEBuilder HiFi DNA Assembly Cloning Kit (NEB, E5520). The mix was transformed to Escherichia coli DH5alpha strain and plated to media supplemented with 100 μg/ml ampicillin. The plates were incubated at 37°C. Eighteen hours after transformation >50 000 colonies were washed off the plates, and the plasmid library was extracted by Qiagen Plasmid Maxi kit (Qiagen 12162). HTS analysis of the library showed representation of 15716 PAM variants. The library plasmid map is presented in the Supplementary Table S1. Competent E. coli Star cells carrying pACYC184_CcCas9_locus or an empty pACYC184 vector were transformed with 7N PAM plasmid libraries and plated to 100 μg/ml ampicillin and 25 μg/ml chloramphenicol containing agar plates. After 16 h, cells were harvested and DNA was extracted using Qiagen Plasmid Maxi kit (Qiagen 12162). PAM-containing sequences were PCR amplified using M13_f and M13_r primers and sequenced using Illumina platform with pair-end 150 cycles (75 + 75).

The minicircle plasmids were produced as described previously6 (link)12 (link). The episomal reprogramming plasmids used for this study were purified using the QIAGEN Plasmid Maxi Kit (QIAGEN, USA). The 4-in-1 CoMiP construct was transformed into NTC4862 DH5α competent cells and then plated on 6% sucrose solid media (see Nature Technology's NTC vector User's Manual) and propagated at 30°C for 24–48 hours. Individual colonies were picked and miniprep was performed (QIAprep Spin Miniprep Kit, USA). Thereafter, a 100 μl aliquot of the 4-in-1 CoMiP bacterial culture was inoculated with 100 ml of 6% sucrose liquid media and incubated for 16–18 hr at 37°C. This culture was then used to isolate the 4-in-1 CoMiP vector by using the QIAGEN Plasmid Maxi Kit.