Journal: bioRxiv

Article Title: Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells

doi: 10.1101/2023.12.13.571376

Figure Lengend Snippet: Two-component vector system where Cas9 is integrated into the AAVS1 safe harbor locus to sustain stable expression. The second component is a lentiviral system containing a sgRNA targeting on and off-target sites downstream of an AcrIIA4 variant that is uniquely barcoded. B. Editing efficiency measured over time within cells expressing wild-type AcrIIA4 (circular points) versus those without wild-type AcrIIA4 (rectangular). C. Indel mutation profiles spanning the on- and off-target regions. Deletions are shown in blue and insertions are in orange. D. Experimental design in which AcrIIA4 variants were delivered to A549 cells expressing Cas9, which were cultured for 10 days prior to collection and sequencing of the AcrobaTx barcode. E. Structure of the AcrobaTx barcode and definitions of gene editing activity metrics derived from measuring editing outcomes within the on- and off-target sites. F. Editing precision of alanine scanning variants normalized to the wild-type control. D69A variant is highlighted as the variant with the highest precision within the alanine scan mutant group. G. Crystal structure of AcrIIA4 bound to the SpCas9-gRNA complex. The interaction between D69 and R1335, required for PAM recognition, is highlighted. H. Distribution of editing efficiency ( EE , left) and editing precision ( EP , right) across all AcrIIA4 variants. Variants that resulted in EP in the top 90 th percentile are highlighted in red. I. Deletion profiles of DNA repair outcomes from cells without AcrIIA4 (blue) versus cells that express a variant that promotes precise editing (red). J. Editing precision measured across AcrIIA4 variants at Day 10 versus at Day 7 (Pearson Correlation Coefficient r = 0.56).

Article Snippet: A549 cells expressing the pAG001 Cas9 vector (Genecopeia) were transduced at high MOI with 8 μg/mL polybrene using a spin-infection at 1200*g for 45 minutes.

Techniques: Plasmid Preparation, Expressing, Variant Assay, Mutagenesis, Cell Culture, Sequencing, Activity Assay, Derivative Assay, Control

Journal: bioRxiv

Article Title: Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells

doi: 10.1101/2023.12.13.571376

Figure Lengend Snippet: A. Experimental design in which the top 2000 AcrIIA4 variants, rank-ordered by EP , were delivered to A549 and 293T cells for another round of screening. B. Distribution of EP across all sample groups in the second round of screening versus the first round, at left. C. Frequency of editing within the AcrobaTx barcode across seven lead candidate enAcr groups. Yellow rectangles indicate areas in which editing occurs frequently while violet depicts areas with low editing activity. D. Ratio of on- to off-target editing in cells expressing seven representative lead candidate enAcrs (purple) versus ratios observed from DNA repair outcomes within cells expressing previously described benchmark AcrIIA4 variants.

Article Snippet: A549 cells expressing the pAG001 Cas9 vector (Genecopeia) were transduced at high MOI with 8 μg/mL polybrene using a spin-infection at 1200*g for 45 minutes.

Techniques: Activity Assay, Expressing