Fig. 2: Cas9 tolerates whole-domain deletions while maintaining target-binding activity.

A In vivo transcription repression activity of MISER-dCas9 variants with specified amino acids deleted, targeting either GFP (left) or RFP (right). dCas9s with REC2, REC3, HNH, or RuvC domain deletions have near-WT binding activity when targeted to GFP. When targeted to RFP, ΔREC2, and ΔREC3 show less robust binding activity. Data are normalized to vector-only control representing maximum fluorescence. Data are plotted as mean ± SD from biological triplicates. B Schema showing cloned MISER constructs with individual domain deletions corresponding to tolerated regions found in MISER screen. C Bio-layer interferometry (BLI) assay of MISER constructs. ΔREC2 and ΔREC3 exhibit weak binding against a fully complementary dsDNA target, while ΔHNH and ΔRuvC show intermediate binding. Binding is rescued to near-WT levels in ΔREC2 and ΔREC3, although at a slower rate, when the dsDNA contains a 3-bp bubble in the PAM-proximal seed region. Data are normalized to dCas9 binding to fully complementary dsDNA. D U-251 cells stably expressing the indicated MISER-dCas9 or WT-dCas9 KRAB fusion. Proteins were transduced with mCherry-tagged lentiviral vectors expressing sgRNAs targeting essential genes (sgPCNA, sgRPA1) or nontargeting controls (sgNT). At Day 2 post transduction, cells were mixed with the respective parental population; mCherry fluorescence was monitored over time. Data represent the mean and SD of triplicates (n = 3). Significance in cell depletion was assessed by comparing samples to their respective Day 2 controls using unpaired, two-tailed t tests (α = 0.01). E Measurement of CRISPRi efficacy of single-deletion MISER constructs in mammalian U-251 cells using RT-qPCR. U-251 cells were stably transduced with lentiviral vectors encoding dCas9 or MISER constructs fused with a KRAB repressor, along with lentivirus expressing sgRNA targeting PCNA. Cells were harvested 2 (left panel) or 5 (right) days post transduction of the sgRNA and assayed for PCNA expression. Bar graphs represent fold change of PCNA expression relative to a nontargeting sgRNA. Data presented as mean and SD (for triplicates where shown). Source data are provided as a Source Data file.