Journal: bioRxiv

Article Title: Directed evolution of compact RNA-guided nucleases for homology–directed repair in mammalian cells

doi: 10.1101/2025.10.27.684765

Figure Lengend Snippet: (a) Schematic of the homology-directed repair (HDR) fluorescent reporter. A protospacer with its protospacer-associated motif (Un1Cas12f1 PAM: TTTA) or transposon-associated motif (ISDra2 TnpB TAM: TTGAT) was cloned upstream of an EGFP gene lacking a start codon. Lentiviral delivery into HEK293T cells generated a non-fluorescent polyclonal cell line. EGFP expression can be restored upon successful HDR following delivery of a nuclease (CasMINI or TnpB), a guide RNA (sgRNA for CasMINI and ωRNA for TnpB), and a donor single-stranded oligodeoxynucleotide (ssODN) containing the start codon. An arrow indicates approximate cut position. BSD -gene for blasticidin resistance, LTR-long terminal repeat; LV-lentiviral. (b) Schematic of the directed evolution selection workflow. CasMINI or TnpB variant libraries were screened in reporter cells by iterative cycles of HDR, fluorescence-activated cell sorting (FACS) enrichment, and library regeneration to select for improved activity. (c) HDR efficiency measured as bulk EGFP signal across four rounds of selection of indicated proteins, normalized to the respective wild-type nuclease. Bars represent the value of a single experimental replicate. (d) Relative frequency of enriched mutations of the original sequences (Ctrl) and after rounds 1, 2, and 4 (“R1”, “R2”, “R4”, respectively) of selection for indicated nucleases. Mutations with >0.3% frequency and >3.75-fold enrichment over the pre-selection libraries are shown. The clusters with enriched mutations are marked. FC, fold-change. (e) Enriched mutations mapped onto the structures of nuclease Un1Ca12f1 (PDB: 7l49), highlighted in teal and nuclease TnpB (PDB: 8H1J) highlighted in orange. Close-up views for mutation cluster 1 are presented for each nuclease. Protein structures were visualized with ChimeraX .

Article Snippet: EFα-TAM/PAM-protospacer-EGFP-PGK-BSD fragment was inserted in a lentiviral backbone (Addgene ID 60954) using PCR and Gibson assembly.

Techniques: Clone Assay, Generated, Expressing, Selection, Variant Assay, Fluorescence, FACS, Activity Assay, Mutagenesis

Journal: bioRxiv

Article Title: Directed evolution of compact RNA-guided nucleases for homology–directed repair in mammalian cells

doi: 10.1101/2025.10.27.684765

Figure Lengend Snippet: (a) HDR efficiencies of 88 CasMINI variants were tested in a lentiviral (LV) format and 158 CasMINI variants tested with plasmid delivery at Target CM1 in HEK293T cells. HDR values were measured as percentage of EGFP-positive cells from BFP/mCherry double positive cells (BFP marks the guide-expressing construct; mCherry marks the nuclease-expressing construct) and normalized to CasMINI. Highlighted variants (teal) were selected for further validation using plasmid delivery in duplicate. (b) HDR efficiencies of single mutants (SMs) derived from selected high-performing CasMINI variants or enriched in NGS after round four of selection, tested at Target CM1 in HEK293T cells. HDR values were measured as percentage of EGFP-positive cells from BFP/mCherry double positive cells (BFP marks the guide-expressing construct; mCherry marks the nuclease-expressing construct) and normalized to CasMINI ( n = 2 biological replicates). Highlighted SMs (teal) were selected for further testing at the orthogonal HDR reporter target (Target CM2). A378V was not selected due to the presence of A378T with similar or slightly higher editing efficiency, which was chosen for further analysis. Each dot represents an individual biological replicate, and bars represent the mean. (c) HDR efficiencies of selected CasMINI SMs tested in parallel at two distinct targets (Target CM1 and Target CM2) in HEK293T cells, measured as percentage of EGFP-positive cells from BFP/mCherry double positive cells (BFP marks the guide-expressing construct; mCherry marks the nuclease-expressing construct), with values normalized to CasMINI ( n = 2 biological replicates). The mutations selected for making combinatorial variants are highlighted. Each dot represents an individual biological replicate, and bars represent the mean. (d) Western blot analysis of CasMINI and CasMINI_CV20 protein expression levels. Expression of each nuclease was performed in triplicate together with sgRNA for Target CM1 in HEK293T cells. GAPDH was used as a loading control. (e) HDR efficiencies of eight newly generated CasMINI variants, each containing one mutation derived from EVOLVEpro, cloned onto the CasMINI_CV20 background, tested in HEK293T cells ( n = 2 biological replicates). HDR values were measured as percentage of EGFP-positive cells from BFP/mCherry double positive cells (BFP marks the guide-expressing construct; mCherry marks the nuclease-expressing construct) and normalized to the previously best-performing variant, CasMINI_CV20. Each dot represents an individual biological replicate, and bars represent the mean. (f) Comparison of editing efficiencies of Un1Cas12f1, CasMINI, Un1Cas12f1_CV20, CasMINI_CV20 at Target CM1 and Target CM2 in HEK293T cells. HDR values were measured as percentage of EGFP-positive cells from BFP/mCherry double positive cells (BFP marks the guide-expressing construct; mCherry marks the nuclease-expressing construct) and normalized to Un1Cas12f1 ( n = 3 biological replicates). Each dot represents an individual biological replicate, and bars represent the mean ± standard deviation. All P values were calculated using an unpaired and two-sided t -test, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: EFα-TAM/PAM-protospacer-EGFP-PGK-BSD fragment was inserted in a lentiviral backbone (Addgene ID 60954) using PCR and Gibson assembly.

Techniques: Plasmid Preparation, Expressing, Construct, Biomarker Discovery, Derivative Assay, Selection, Western Blot, Control, Generated, Mutagenesis, Clone Assay, Variant Assay, Comparison, Standard Deviation