Walton Russell T, Qin Yue, Blainey Paul C
Broad Institute of MIT and Harvard, Cambridge, MA, USA.
Department of Biological Engineering, MIT, Cambridge, MA, USA.
bioRxiv. 2024 Mar 17:2024.03.17.585235. doi: 10.1101/2024.03.17.585235.
Forward genetic screens seek to dissect complex biological systems by systematically perturbing genetic elements and observing the resulting phenotypes. While standard screening methodologies introduce individual perturbations, multiplexing perturbations improves the performance of single-target screens and enables combinatorial screens for the study of genetic interactions. Current tools for multiplexing perturbations are incompatible with pooled screening methodologies that require mRNA-embedded barcodes, including some microscopy and single cell sequencing approaches. Here, we report the development of , a CROPseq-inspired lentiviral system to multiplex (Sp) Cas9-based perturbations with mRNA-embedded barcodes. CROPseq-multi has equivalent per-guide activity to CROPseq and low lentiviral recombination frequencies. CROPseq-multi is compatible with enrichment screening methodologies and optical pooled screens, and is extensible to screens with single-cell sequencing readouts. For optical pooled screens, an optimized and multiplexed detection protocol improves barcode detection efficiency 10-fold, enables detection of recombination events, and increases decoding efficiency 3-fold relative to CROPseq. CROPseq-multi is a widely applicable multiplexing solution for diverse SpCas9-based genetic screening approaches.
正向遗传筛选旨在通过系统地扰动遗传元件并观察由此产生的表型来剖析复杂的生物系统。虽然标准筛选方法引入单个扰动，但多重扰动可提高单靶点筛选的性能，并能进行组合筛选以研究基因相互作用。当前用于多重扰动的工具与需要mRNA嵌入条形码的汇集筛选方法不兼容，包括一些显微镜和单细胞测序方法。在此，我们报告了一种受CROPseq启发的慢病毒系统的开发，该系统可将基于（Sp）Cas9的扰动与mRNA嵌入条形码进行多重整合。CROPseq-multi与CROPseq具有相同的单向导活性，且慢病毒重组频率较低。CROPseq-multi与富集筛选方法和光学汇集筛选兼容，并且可扩展到具有单细胞测序读数的筛选。对于光学汇集筛选，一种经过优化的多重检测方案将条形码检测效率提高了10倍，能够检测重组事件，并且相对于CROPseq，解码效率提高了3倍。CROPseq-multi是一种广泛适用于多种基于SpCas9的基因筛选方法的多重整合解决方案。
