Oligonucleotides and gblocks

The starting plasmid pDsRed (Addgene plasmid #51019) was provided by Melissa Harrison, Kate O'Connor-Giles, and Jill Wildonger, pnos-Cas9-nos (Port et al., 2014 (link); Addgene plasmid #62208) was provided by Simon Bullock, and VP12 (Kleinstiver et al., 2016 (link); Addgene plasmid #72247) was provided by Simon Bullock. Starting plasmids ATSacG, TTTgRNAtRNAi, TTTgRNAt, BHDgN1c, and BHDgN1cv3 were constructed in a previous study (Champer et al., 2020c (link)). Restriction enzymes for plasmid digestion, Q5 Hot Start DNA Polymerase for PCR, and Assembly Master Mix for Gibson assembly were acquired from New England Biolabs. Oligonucleotides and gBlocks were obtained from Integrated DNA Technologies. JM109 competent cells and ZymoPure Midiprep kit from Zymo Research were used to transform and purify plasmids. Cas9 gRNA target sequences were identified by the use of CRISPR Optimal Target Finder (Gratz et al., 2014 (link)). A list of DNA fragments, plasmids, primers, and restriction enzymes used for cloning each construct can be found in Supplementary file 3. The annotated sequences of the final construct insertion plasmids can be found in Supplementary file 4 (ApE format, http://biologylabs.utah.edu/jorgensen/wayned/ape).

Recombinant DNA methods were in accordance with established procedures and used commercially available reagents: Phusion High-Fidelity DNA Polymerase (Thermo Fisher, Waltham, MA); restriction enzymes and β-agarase (New England BioLabs, Beverly, MA); T4 DNA ligase, CIP, and T4 PNK (Roche, Nutley, NJ); GTG low melting temperature agarose for in-gel cloning, (Lonza, Walkersville, MD); oligonucleotides and gBlocks® (Integrated DNA Technologies, Coralville, IA); Assemblies involving polymerase chain reaction (PCR) amplification were sequenced through the inserts and junctions to verify the desired construct. Cloning was carried out in XL1-Blue cells. Full details of cloning, oligonucleotides, maps, and sequences of the resulting constructs are available on request.