u6 sgrna cassette  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: A side-by-side comparison of variant function measurements using deep mutational scanning and base editing

doi: 10.1093/nar/gkaf738

Figure Lengend Snippet: Adenosine base editor screen of full length of BCR-ABL. ( A ) Schematic of adenosine base editor screen. Three days after infection with BCR-ABL sgRNA library, Ba/F3 EGFP-P2A-BCR-ABL ABE8e cells were selected with 1 mg/ml hygromycin for 6 days and pelleted. Guides were PCR-amplified and sequenced. ( B ) A sliding window analysis using a window size of 40 sgRNAs. In a window we quantified the proportion of BCR-ABL sgRNAs that drop out more extreme than a Z-score of −4 of the negative control sgRNA growth rate ( N = 3). ( C ) Correlation of the same ABE BCR-ABL screens performed in K562s ( N = 2) and Ba/F3 expressing BCR-ABL ( N = 3). ( D ) Kernel density estimate of growth rate distributions of non-targeting control, and BCR-ABL sgRNA libraries. Dashed gray line represents a –2 Z-score of the targeting control. ( E ) Lollipop plot displays dropout of each sgRNA across the ABL1 kinase domain. Dashed gray line represents a –2 Z-score of the targeting control ( N = 3).

Article Snippet: ABE8e SpG plasmid was made by deleting the U6 sgRNA cassette from pRDA_479 (Addgene #179099) [ ] using NEB KLD (NEB #M0554S).

Techniques: Infection, Amplification, Negative Control, Expressing, Control

Journal: Nucleic Acids Research

Article Title: A side-by-side comparison of variant function measurements using deep mutational scanning and base editing

doi: 10.1093/nar/gkaf738

Figure Lengend Snippet: Comparison of adenosine base editor sgRNA growth rate and their respective mutation growth rates from DMS. Each dot represents a mutation an sgRNA is predicted to make. Dashed lines represent –2 Z-score of the non-deleterious distribution, and negative control sgRNA for the DMS and ABE screens, respectively. These cutoffs are used to define if an sgRNA or mutation is deleterious. If an sgRNA and its mutation do not deplete in their respective screen, in other words, both are non-deleterious, then they are colored yellow. If they both are deleterious, or true positive, then they are colored orange. If an sgRNA depletes, but the predicted edit does not deplete, a false positive, then the dot is colored in the blue. If a sgRNA fails to deplete, and the predicted mutation(s) are deleterious, a false negative, then that point is colored in green. ( A ) Shows all possible edits between nucleotides 2 and 12. ( B ) Shows only the most likely edits, those between nucleotides 4 and 8. ( C ) Shows only sgRNA predicted to be efficient, and edits between nucleotides 4 and 8. ( D ) Shows sgRNAs that are predicted to make only a single edit between nucleotides 4 and 8. ( E ) The distribution of edits can be estimated by machine learning model called BE-HIVE. ( F ) Correlation between predicted sgRNA growth rate and observed sgRNA growth rate. The x -axis shows the predicted growth rate of each sgRNA based on a weighted sum of the probability edit(s), and the effect of that edit(s) from DMS data. The y -axis shows the measured growth rate of the efficiently editing sgRNAs from the ABE screen.

Article Snippet: ABE8e SpG plasmid was made by deleting the U6 sgRNA cassette from pRDA_479 (Addgene #179099) [ ] using NEB KLD (NEB #M0554S).

Techniques: Comparison, Mutagenesis, Negative Control

Journal: Nucleic Acids Research

Article Title: A side-by-side comparison of variant function measurements using deep mutational scanning and base editing

doi: 10.1093/nar/gkaf738

Figure Lengend Snippet: Medium-throughput pooled adenosine base editor screen. ( A ) Schematic of medium-scale validations screen of 71 sgRNAs targeting ABL1 kinase, where the edits and sgRNA are sequenced after IL-3 withdrawal. (B, C) Growth rates of sgRNA-induced edits. Each dot represents a specific edit and its measured growth rate, while each “X” or filled circle indicates whether the corresponding mutation was deleterious or nondeleterious in a prior DMS experiment. ( B ) Growth rates for all detected edits within the 4–8 nucleotide editing window of their respective sgRNAs. Gray lines connect edits generated by the same sgRNA. ( C ) Highlights the most prevalent edits, defined as those occurring at a frequency of over 50% of all edits within the sgRNA’s editing window. ( D ) Weighted model for sgRNA growth rate. The sgRNA growth rate was predicted by a weighted sum of the growth rates of all edits within its 4–8 bp editing window, with weights corresponding to the frequency of each edit. The black line indicates a perfect correlation between the predicted and experimentally measured sgRNA growth rates. (E–G) Comparison of growth rates from the pooled ABE screen and DMS data. These panels directly compare the growth rates of specific amino acid mutants measured in the pooled ABE screen and the prior DMS experiment. The amino acid (AA) count represents the number of amino acids simultaneously edited. ( E ) All edits detected within the 4–8 bp editing window. ( F ) Highlights high-confidence growth rate measurements by applying a stringent edit frequency cutoff of 0.01, and ( G ) focuses exclusively on single amino acid edits to enable a direct comparison between the ABE screen and DMS data ( N = 3).

Article Snippet: ABE8e SpG plasmid was made by deleting the U6 sgRNA cassette from pRDA_479 (Addgene #179099) [ ] using NEB KLD (NEB #M0554S).

Techniques: Mutagenesis, Generated, Comparison