Gblock

pETM6 and pETM6-mCherry were a gift from Mattheos Koffas (Addgene plasmids #49795 and #66534). pLysS was obtained from NEB. Plasmids made in this study were constructed using G-blocks™ and/or PCR products and assembled using NEBuilder® HiFi DNA Assembly Master Mix following manufacturer’s protocol (NEB). PCRs were performed with Q5 DNA Polymerase (NEB). pSMART-HC-Kan (Lucigen, WI), pTWIST-Chlor-Medium Copy, pTWIST-Kan-High Copy (Twist Biosciences, San Francisco, CA), and pCDF (derived from pCDF-1b; EMD Millipore, Burlington, MA) were used as a backbone vectors in these studies. Sequences of all oligos and synthetic DNA are given in Supporting Information Materials Section 3, Tables S3.1 and S3.2. All plasmid sequences were confirmed by DNA sequencing (Genewiz). Sequences and maps are available with Addgene (refer to Table 1 for Addgene numbers; refer to Supporting Information Materials Section).

sgRNAs (oligonucleotide sequences are indicated in Table S3) were cloned into lentiCRISPR-v1 or -v2 linearized with BsmBI by T4 ligase (NEB). sgRNA expressing vector along with lentiviral packaging vectors Delta-VPR and CMV VSV-G were transfected into HEK-293T cells using the XTremeGene 9 transfection reagent (Roche). Similarly, for overexpression cell lines, gBlocks (IDT) containing the cDNA of interest were cloned into pMXS linearized with BamHI and NotI by Gibson Assembly (NEB). cDNA vectors along with retroviral packaging vectors gag-pol and CMV VSV-G were transfected into HEK-293T cells. For FLAG and HA tagged proteins, tags were added to the C terminus of each protein. Media was changed 24 hrs after transfection. The virus-containing supernatant was collected 48 hrs after transfection and passed through a 0.45 μm filter to eliminate cells. Target cells in 6-well tissue culture plates were infected in media containing 8 μg/mL of polybrene and a spin infection was performed by centrifugation at 2,200 rpm for 1 hour. Post-infection, virus was removed and cells were selected with puromycin or blasticidin. For knockout cells, after selection, cells were single-cell diluted into the wells of a 96-well plate. Cells were grown for two weeks, and the resultant colonies were expanded. Clones were validated for loss of the relevant protein via immunoblotting.

Tethered sequences were placed at the 3’-end of the sgRNA sequence (taken from pLKO.1-puro U6 sgRNA), separated by a spacer. The sequences were synthesized as gBlocks (Integrated DNA Technologies (IDT)) that also comprised BfuAI-stuffer (taken from pLKO.1-puro U6 sgRNA) and a Pol III T₆ terminator (sequences in Supplementary Table 2). The gBlocks were digested with AgeI and EcoRI (New England Biolabs) and ligated into pLKO.1-puro U6 sgRNA BfuAI stuffer (Addgene plasmid #50920, a gift from Rene Maehr and Scot Wolfe ^{64 (link)}).
The single guide RNA (sgRNA) targeting sequence for Sorcs2 was previously described ^{41 (link)}. Other sgRNAs were designed using CHOPCHOPv2 ^{65 (link)} (sequences in Supplementary Table 2), synthesized as oligonucleotides, annealed, and inserted into the vector using the BfuAI site.
A G-rich sequence with the same G-content as the tethered G-tract sequence, appended to the Fgf11 sgRNA and a 5’ spacer sequence, was ordered as a gBlock (sequence in Supplementary Table 2), digested with NdeI and EcoRI and cloned into pLKO.1-puro U6 sgRNA BfuAI stuffer.
A G-tract RNA sequence spanning the Fgf11 exon-intron junction (chr11:69,801,412-69,801,633 in mm10), appended to the Fgf11 sgRNA and a 5’ spacer sequence, was ordered as a gBlock (sequence in Supplementary Table 2), digested with NdeI and EcoRI and cloned into pLKO.1-puro U6 sgRNA BfuAI stuffer.

sgRNAs (oligonucleotide sequences are indicated in Table S3) were cloned into lentiCRISPR-v2 linearized with BsmBI by T4 ligase (NEB). sgRNA expressing vector along with lentiviral packaging vectors Delta-VPR and CMV VSV-G were transfected into HEK-293T cells using the XTremeGene 9 transfection reagent (Roche). Similarly, for overexpression cell lines, gBlocks (IDT) containing the cDNA of interest were cloned into pMXS linearized with BamHI and NotI by Gibson Assembly (NEB). cDNA vectors along with retroviral packaging vectors gag-pol and CMV VSV-G were transfected into HEK-293T cells. The virus-containing supernatant was collected 48 hrs after transfection and passed through a 0.22 μm filter to eliminate cells. Target cells in 6-well tissue culture plates were infected in media containing 8 μg/mL of polybrene and a spin infection was performed by centrifugation at 2,200 rpm for 1 hour. Post-infection, virus was removed and cells were selected with puromycin or blasticidin. For Atg7 knockout cells, after selection, cells were single-cell diluted into the wells of a 96-well plate. Cells were grown for two weeks, and the resultant colonies were expanded. Clones were validated for loss of the relevant protein via immunoblotting.