Scalable single-cell pooled CRISPR screens with conventional knockout vector libraries

Islam, M.; Yang, Y.; Simmons, A. J.; Xu, Y.; Fisher, E.; Deng, W.; Grieb, B.; Molina, P.; Caestecker, C. d.; Ramirez-Solano, M. A.; Liu, Q.; Tansey, W.; Macara, I. G.; Rathmell, J.; Coffey, R. J.; Lau, K.

doi:10.1101/2024.02.01.578192

bioRxiv · 10.1101/2024.02.01.578192

Scalable single-cell pooled CRISPR screens with conventional knockout vector libraries

Islam, M.·Yang, Y.·Simmons, A. J.·Xu, Y.·Fisher, E.·Deng, W.·Grieb, B.·Molina, P.·Caestecker, C. d.·Ramirez-Solano, M. A.·Liu, Q.·Tansey, W.·Macara, I. G.·Rathmell, J.·Coffey, R. J.·Lau, K.

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Abstract

Current methods for single-cell RNA profiling of pooled CRISPR screens are limited, either by indirect capture of single guide RNAs (sgRNAs) or by custom modification of plasmid libraries. Here, we present a direct sgRNA capture platform called Native sgRNA Capture and sequencing (NSC-seq) that enables single-cell CRISPR screens using common knockout plasmid libraries, facilitating genotype-phenotype mapping at multiple scales in vitro and in vivo. Additionally, we characterize sgRNA expression in three whole-genome knockout libraries, revealing a substantial subset of truncated (isoform) spacer reads. We provide this dataset as a reference of expressed sgRNA isoforms that may potentially have compromised CRISPR gene editing efficacy and precision.

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Islam, M., Yang, Y., Simmons, A. J., Xu, Y., Fisher, E., Deng, W., Grieb, B., Molina, P., Caestecker, C. d., Ramirez-Solano, M. A., Liu, Q., Tansey, W., Macara, I. G., Rathmell, J., Coffey, R. J., Lau, K.. 2024-02-02. Scalable single-cell pooled CRISPR screens with conventional knockout vector libraries. https://doi.org/10.1101/2024.02.01.578192

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