MISER library construction: plasmid recombineering

Two sets of 1368 oligonucleotides were designed and ordered as Oligonucleotide Library Synthesis from Agilent Technologies (Table ^S1). Oligonucleotides were designed to insert a six base-pair (bp) recognition sequence for either the restriction enzyme NheI or SpeI between every codon in dCas9 (Supplementary Fig. ^1A). The full list of ordered oligonucleotides is available as Auxiliary Supplementary Materials—Recombineering Oligonucleotides. Internal priming sites were included to amplify NheI- or SpeI-specific oligonucleotide libraries. A modified amplification procedure was performed as follows. In a 50 μL PCR reaction, 10 ng of template oligonucleotide library was amplified according to the manufacturer’s instructions, but with an extension time of only 5 s, and a total of only 15 cycles. Dimethyl sulfoxide (1.5%) was also included in the PCR reaction. These modifications were empirically determined to minimize undesirable higher-order PCR products that were observed to be produced by amplification. These side products are likely the result of complementary oligonucleotides priming one another. Notably, this phenomenon is likely inherent to the amplification of a library of DNA tiled across a common sequence—in this case dCas9. PCR primers can be found in Table ^S6 and Auxiliary Supplementary Materials—Primer Sequences. Twenty-four such reactions were typically performed in parallel and then combined, followed by concentration with Zymo DNA Clean & Concentrator. BsmbI restriction digestion was then used to remove priming ends, followed by a second concentration with Zymo DNA Clean & Concentrator, resulting in mature double-stranded recombineering-competent DNA.

Plasmid recombineering was performed as described in Higgins et al.³⁸, using strain EcNR2 (Addgene, ID: 26931) to generate MISER libraries in plasmid pSAH060. Plasmid sequences can be found in Auxiliary Supplementary Materials—Plasmid Sequences. Briefly, mature double-stranded recombineering-competent DNA at a final volume of 50 μL of 1 μM, plus 10 ng of pSAH060, was electroporated into 1 mL of induced and washed EcNR2 using a 1 mm electroporation cuvette (Bio-Rad GenePulser). A Harvard Apparatus ECM 630 Electroporation System was used with settings 1800 kV, 200 Ω, 25 μF. Three replicate electroporations were performed, and then individually allowed to recover at 30 °C for 1 h in 1 mL of SOC (Teknova) without antibiotic. LB (Teknova) and kanamycin (Fisher) at 60 μg/mL were then added to 6 mL final volume and grown overnight. A sample of recovered culture was diluted and plated on kanamycin to estimate the total number of transformants, typically >10⁷. Cultures were miniprepped and combined the next day. Plasmid recombineering is relatively inefficient, and only a fraction of recovered plasmids contained successful NheI or SpeI insertions. To recover completely penetrant libraries, an intermediate cloning step was performed. A PCR product conferring resistance to chloramphenicol was cloned into both libraries of pSAH060 plasmids (Auxiliary Supplementary Materials—Chloramphenicol Selection). This PCR product contained either flanking NheI restriction sites or SpeI restriction sites, such that only modified pSAH060 plasmids (possessing NheI or SpeI restriction sites) could obtain chloramphenicol resistance through NheI/SpeI digestion and subsequent ligation. Libraries were then purified (Zymo) and transformed into XLI-Blue-competent cells for overnight selection in chloramphenicol (Amresco) at 25 μg/mL, followed by plasmid isolation the next day. Samples of recovered cultures were also plated on both kanamycin alone (native pSAH060 resistance) and chloramphenicol alone (resistance mediated by successful recombineering insertion) to estimate the fraction of modified plasmids and therefore the restriction library size. Recombineering efficiencies were observed at ~0.5% by this method, indicating restriction library sizes of ~50,000, well above the number of unique insertion sites per library (1368). Finally, chloramphenicol-resistant pSAH060 libraries were digested with either NheI or SpeI as appropriate, removing the chloramphenicol cassette. The libraries were run on an agarose gel, and the 5953 bp (5947 bp pSAH060 + 6 bp inserted restriction site) linear band corresponding to each library was gel extracted. To construct deletion variants composed of N- and C-terminal dCas9 fragments, 1  μg of each library was mixed and digested with BsaI, then cleaned up (Zymo). The resulting DNA mixture contained equimolar free dCas9 N- and C-terminal fragments, as well as an equimolar pSAH060 vector backbone. This mixture was then ligated in the presence of SpeI and NheI, “locking” dCas9 fragments together by one of two 6-bp scar sites not recognized by either enzyme (Supplementary Fig. ^1B). The ligated MISER library was transformed into XL1-Blue, grown overnight, and plasmids were isolated the next day. The MISER library of dCas9 is quite large, with 936,396 possible deletions (N (N + 1)/2, N = 1368), and all cloning steps were performed with validation that >10⁷ transformants were obtained.