Gibson assembly

The pCMVSau plasmid expressing a human codon optimized SaCas9 and a customizable U6-driven gRNA scaffold have been previously described [18 (link)]. Cognate luciferase indicator constructs were generated as previously described [14 (link)]. Maps of these plasmids and all other SaCas9 plasmids are shown in Figure S1 in Additional file 1.
gRNA used in Fig. 1a was generated by cloning annealed oligos containing the target sequence into pCMVSau. gRNAs used for data shown in Figs. 1b–d and 2d were generated by PCR and transfected as amplicons containing U6 promoter, spacer sequence, and TRACR scaffold. gRNAs used for data shown in Figs. 2b, c and 4a, b were generated by ligating either one or two of these into a pUC19 backbone vector via Gibson Assembly (New England Biolabs).
AAV vectors used in Fig. 3a–c were constructed by Gibson Assembly of one or two gRNA cassettes into SaCas9-containing AAV backbone pSS3. Vectors used in Fig. 3d–f were constructed by subcloning gRNA cassette pairs from vectors pAF089, pAF091, pAF092 into pSS60. Inverted terminal repeats (ITRs) were confirmed by XmaI digest of the vectors.

The myo1Cb ORF was amplified from a mixed stage pool of cDNAs using the primers: 5′- GATCCCATCGATTCGACGGAATCCGGGTCATGATG-3′ and 5′-AGGCTCGAGAGGCCTTGGTCACGACTCGTCCATC-3′ and cloned into the pCS2+ vector. Bold letterings indicate primer overhangs that are also present in the pCS2+ sequence and were used to recombine insert and vector using Gibson assembly (NEB). A similar strategy was used to clone the myo1D ORF into pCS2+ using the primers 5′-GATCCCATCGATTCGACAGCTTATTATGGCAGAACACG-3′ and 5′-AGGCTCGAGAGGCCTTAAATGGGATCCTGGTCCTCTAG-3′.
To test the potential residual activity of the Myo1D^tj16b mutant protein, the tj16b mutation was introduced into myo1D-pCS2+ through amplification with the primers 5′-GCATAATGTATTTACTAGCCTTTTTCCAGCTCCACTCTCCC-3′ and 5′-GGCTAGTAAATACATTATGC-3′, followed by re-ligation through Gibson assembly (NEB).

The two-plasmid bacterial CRISPRi system pdCas9-bacteria_GNE and PGNRNA-bacteria_GNE are based on the AddGene plasmids 44249 and 44251 (73 (link)), respectively. The plasmid was synthesized in smaller DNA fragments (500 bp to 3 kb) (IDT gBlocks) and assembled by Gibson assembly (NEB) according to the manufacturer’s protocols. Plasmids were confirmed by sequencing (ELIM Bio). gRNAs were designed to target the 5′ end of the gene on the nontemplate strand using Benchling CRISPR software (74 (link)). gRNAs were cloned into PGNRNA-bacteria using Gibson assembly (NEB) according to the manufacturer’s protocols and sequence confirmed (ELIM Bio).
Bacterial cultures were grown overnight on LB agar supplemented with carbenicillin (50 μg/ml) and chloramphenicol (12.5 μg/ml) to maintain both plasmids, pdCas9-bacteria and PGNRNA-bacteria with each gRNA as appropriate. Cells were scraped from the plate into fresh medium. The OD₆₀₀ was measured, and the culture was subsequently diluted to an OD₆₀₀ 0.001 in the presence or absence of G2824, globomycin, or ciprofloxacin. Two hundred microliters was transferred to a 96-well plate (Corning) and monitored for growth by measuring the OD₆₀₀ (EnVision multimode plate reader; PerkinElmer). All treatments were performed in triplicates. The specificity of CRISPRi downregulation was measured using reverse transcriptase quantitative PCR (RT-qPCR).

Base editor plasmids were constructed by replacing deaminase and Cas-protein domains of the p2T-CMV-ABE7.10-BlastR (Addgene 152989) plasmid by USER cloning (New England Biolabs)(50 (link)). Individual sgRNAs were cloned into the SpCas9-hairpin U6 sgRNA expression plasmid (Addgene 71485) using BbsI plasmid digest and Gibson assembly (New England Biolabs). Protospacer sequences and gene-specific primers used for amplification followed by HTS are listed in Supplementary Table 1. Constructs were transformed into Mach1 chemically competent E. coli (ThermoFisher) grown on LB agar plates and liquid cultures were grown in LB broth overnight at 37 °C with 100 μg/mL ampicillin. Individual colonies were validated by Templiphi rolling circle amplification (ThermoFisher) followed by Sanger sequencing. Verified plasmids were prepared by mini, midi, or maxiprep (Qiagen).
AAV vectors were cloned by Gibson assembly (NEB) using NEB Stable Competent E. coli (High Efficiency) to insert the sgRNA sequence and C-terminal base editor half of ABE8e-SpyMac into v5 Cbh-AAV-ABE-NpuC+U6-sgRNA (Addgene 137177), and the N-terminal base editor half and a second U6-sgRNA cassette into v5 Cbh-AAV-ABE-NpuN (Addgene 137178)(74 (link)).

The pCDNA3 mito-APEX plasmid was published previously (Rhee et al., 2013 (link)). The Mito-APEX2 construct was cloned from this plasmid using a two-step protocol. First, the A134P mutation (Lam et al., 2015 (link)) was introduced into the APEX gene itself, using QuikChange mutagenesis (Agilent Technologies, Santa Clara, CA), and thereafter the APEX2 gene was moved to the lentiviral vector pLX304, via Gateway cloning (ThermoFisher Scientific, Waltham, MA), to generate the plasmid pLX304 mito-APEX2. Other APEX-fusion constructs (pLX304 APEX2-NLS, pLX304 APEX2-NES, and pLX304 ERM-APEX2) were cloned by Gibson assembly (New England Biolabs, Ipswich, MA), using PCR to add targeting sequences and Gibson assembly homology arms to the APEX2 gene, and joining the resulting insert into the pLX304 vector digested by BstBI and NheI. To clone HRP-KDEL, the HRP-KDEL-IRES-Puromycin cassette from HRP C (Martell et al., 2016 (link)), was PCR-amplified and cloned into pCDNA3 using NotI and XbaI sites. Targeting sequences and restriction sites for all constructs are listed in (Supplementary file 5A).