Viswanatha Raghuvir, Entwisle Samuel, Hu Yanhui, Kim Ah-Ram, Reap Kelly, Butnaru Matthew, Qadiri Mujeeb, Mohr Stephanie E, Perrimon Norbert
Department of Genetics, Harvard Medical School, Boston, MA, 02115, USA.
Howard Hughes Medical Institute, Boston, MA, 02115, USA.
Nat Commun. 2025 Jul 15;16(1):6498. doi: 10.1038/s41467-025-61692-3.
CRISPR screens enable systematic, scalable genotype-to-phenotype mapping. We previously developed a CRISPR screening method for Drosophila melanogaster and mosquito cell lines using plasmid transfection and site-specific integration to introduce single guide (sgRNA) libraries. The method relies on weak sgRNA promoters to avoid early CRISPR-Cas9 activity causing discrepancies between genome edits and integrated sgRNAs. To address this issue and utilize higher strength sgRNA expression, we introduce a method to co-transfect a plasmid expressing anti-CRISPR protein to suppress early CRISPR-Cas9 activity which we term "IntAC" (integrase with anti-CRISPR). IntAC dramatically improves precision-recall of fitness genes across the genome, allowing us to generate the most comprehensive list of cell fitness genes yet assembled for Drosophila. Drosophila fitness genes show strong correlation with human fitness genes and underscore the effects of paralogs on gene essentiality. We also perform a resistance screen to proaerolysin, a glycosylphosphatidylinositol-(GPI)-binding pore-forming toxin, retrieving 18/23 expected and one previously uncharacterized GPI synthesis gene. We also demonstrate that an IntAC sublibrary enables precise positive selection of a transporter under solute overload. IntAC represents a straightforward enhancement to existing Drosophila CRISPR screening methods, dramatically increasing accuracy, and might also be broadly applicable to virus-free CRISPR screens in other cell and species types.
CRISPR筛选能够实现系统、可扩展的基因型到表型的映射。我们之前开发了一种针对黑腹果蝇和蚊子细胞系的CRISPR筛选方法，该方法利用质粒转染和位点特异性整合来引入单向导RNA（sgRNA）文库。该方法依赖于弱sgRNA启动子，以避免早期CRISPR-Cas9活性导致基因组编辑与整合的sgRNA之间出现差异。为了解决这个问题并利用更高强度的sgRNA表达，我们引入了一种共转染表达抗CRISPR蛋白的质粒以抑制早期CRISPR-Cas9活性的方法，我们将其称为“IntAC”（整合酶与抗CRISPR）。IntAC显著提高了全基因组适应性基因的精确召回率，使我们能够生成迄今为止为果蝇组装的最全面的细胞适应性基因列表。果蝇适应性基因与人类适应性基因显示出强烈的相关性，并强调了旁系同源基因对基因必需性的影响。我们还对气单胞菌溶素（一种糖基磷脂酰肌醇（GPI）结合的成孔毒素）进行了抗性筛选，找回了23个预期基因中的18个以及一个先前未表征的GPI合成基因。我们还证明了一个IntAC子文库能够在溶质过载的情况下对一种转运蛋白进行精确的正向选择。IntAC是对现有果蝇CRISPR筛选方法的一种直接改进，显著提高了准确性，并且可能也广泛适用于其他细胞和物种类型的无病毒CRISPR筛选。
