Uncoupling of SgRNAs from Their Associated Barcodes During PCR Amplification of Combinatorial CRISPR Screens

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2018 May 26

PMID 29799876

Citations 28

Authors

Mudra Hegde

Christine Strand

Ruth E Hanna

John G Doench

Affiliations

Soon will be listed here.

Abstract

Many implementations of pooled screens in mammalian cells rely on linking an element of interest to a barcode, with the latter subsequently quantitated by next generation sequencing. However, substantial uncoupling between these paired elements during lentiviral production has been reported, especially as the distance between elements increases. We detail that PCR amplification is another major source of uncoupling, and becomes more pronounced with increased amounts of DNA template molecules and PCR cycles. To lessen uncoupling in systems that use paired elements for detection, we recommend minimizing the distance between elements, using low and equal template DNA inputs for plasmid and genomic DNA during PCR, and minimizing the number of PCR cycles. We also present a vector design for conducting combinatorial CRISPR screens that enables accurate barcode-based detection with a single short sequencing read and minimal uncoupling.

Citing Articles

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