column purification  (New England Biolabs)

Journal: bioRxiv

Article Title: The Clearance of Human Cytomegalovirus Using CRISPR/Cas9 RNA Lipid Nanoparticles

doi: 10.64898/2025.12.17.694960

Figure Lengend Snippet: (a) The mechanism of clearing HCMV infections using CRISPR/Cas9 RNA LNPs. (b) Viral gene function categorization. Viral genes were systematically groupedbased on function. (c) Next, all available nucleotide sequences of the categorized viral genes were aligned to identify conserved nucleotides (defined as ≥95% consensus among all input sequences). (d) Conserved CRISPR/Cas9 targets, defined as a (i) 20-nucleotide region with (ii) ≥95% consensus across all input sequences and (iii) an appropriate protospacer adjacent motif, that had optimal on- and off-target scores were selected. (e) GO: biological processes with the highest number of associated genes (top 5, left graph) and the number of essential genes in each category (right graph). Viral genes found under bidirectional double-stranded viral DNA replication classification were selected. (f) Nucleotide conservation plot of selected viral gene. The percent (%) conservation at a given nucleotide position was determined by DNA alignment (MAFFT) of approximately ∼346 ± 6 sequences. A conserved targetable site can be found on both the sense (blue) and anti-sense (purple) strand. The pink line depicts the location of the chosen CRISPR target.

Article Snippet: In vitro transcribed Cas9 mRNA was purified using RNA cleanup spin columns (New England Biolabs, T2050), resuspended in nuclease-free water, and stored in −80 °C until use.

Techniques: CRISPR