CloneSifter: enrichment of rare clones from heterogeneous cell populations
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RESEARCH ARTICLE
Open Access
CloneSifter: enrichment of rare clones from heterogeneous cell populations David Feldman1,2†, FuNien Tsai1,3†, Anthony J. Garrity1,4, Ryan O’Rourke1,5,6, Lisa Brenan1, Patricia Ho1,6, Elizabeth Gonzalez1,6, Silvana Konermann7, Cory M. Johannessen1,8*, Rameen Beroukhim1,9,10*, Pratiti Bandopadhayay1,6,11* and Paul C. Blainey1,12,13*
Abstract Background: Many biological processes, such as cancer metastasis, organismal development, and acquisition of resistance to cytotoxic therapy, rely on the emergence of rare sub-clones from a larger population. Understanding how the genetic and epigenetic features of diverse clones affect clonal fitness provides insight into molecular mechanisms underlying selective processes. While large-scale barcoding with NGS readout has facilitated cellular fitness assessment at the population level, this approach does not support characterization of clones prior to selection. Single-cell genomics methods provide high biological resolution, but are challenging to scale across large populations to probe rare clones and are destructive, limiting further functional analysis of important clones. Results: Here, we develop CloneSifter, a methodology for tracking and enriching rare clones throughout their response to selection. CloneSifter utilizes a CRISPR sgRNA-barcode library that facilitates the isolation of viable cells from specific clones within the barcoded population using a sequence-specific retrieval reporter. We demonstrate that CloneSifter can measure clonal fitness of cancer cell models in vitro and retrieve targeted clones at abundance as low as 1 in 1883 in a heterogeneous cell population. Conclusions: CloneSifter provides a means to track and access specific and rare clones of interest across dynamic changes in population structure to comprehensively explore the basis of these changes. Keywords: Cellular heterogeneity, Barcode targeting, Viable clone-specific cells recovery, Clonal fitness tracking, CRISPR sgRNA-barcode DNA library
Background The response of a heterogeneous population to selection pressure is shaped by the growth dynamics of individual clones within the population. Rare clones can play a decisive role in the outcome of selection. Examples include evasion of antiretroviral therapy by rare HIV variants [1], expansion of drug-resistant cancer cells under chemotherapy [2], and seeding of metastases by clonal tumor cells [3, 4]. In addition, * Correspondence: [email protected]; [email protected]; [email protected]; [email protected] † David Feldman and Fu Nien Tsai contributed equally to this work. 1 Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA Full list of author information is available at the end of the article
comparison of such selected clones with low-fitness clones that perished under selection is likely to provide further insight. Studying how genetic and epigenetic differences affect the survival or disappearance of individual clones during selection provides an op
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