CloneSifter: enrichment of rare clones from heterogeneous cell populations

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.

SunCatcher: Clonal Barcoding with qPCR-Based Detection Enables Functional Analysis of Live Cells and Generation of Custom Combinations of Cells for Research and Discovery

“…A number of innovative barcoding technologies have been developed that enable one to study clonal dynamics among heterogeneous populations of cells across a variety of applications [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] . Many original barcoding methods rely on infecting heterogenous populations of cells with libraries of 10 6 -10 8 barcodes without the ability to isolate and study every individual clone.…”

“…While such high-complexity barcode libraries enable longitudinal quantification of clonal diversity in various experimental settings, they do not enable characterization, functional analyses, or manipulation of the cellular populations of interest. More recently, a number of elegant and sophisticated methods have been developed to trace as well as study barcoded cells that comprise tumors and contribute to disease progression and therapeutic resistance [30][31][32][33][34] .…”

A Cell Double-Barcoding System for Quantitative Evaluation of Primary Tumors and Metastasis in Animals That Uncovers Clonal-Specific Anti-Cancer Drug Effects

“…Previously monitoring cancer development with cell barcoding was done using a barcode library with random barcodes that allows individual labeling of clones within the cancer cell population [6,11,15,17,18]. Since this approach did not allow quantitative comparison of clonal sizes between animals (see Introduction), we realized the necessity of internal controls for normalization.…”

“…Cell DNA barcoding allows dissecting many aspects of tumor dynamics, metastasis and drug effects [4][5][6][7][8][9][10][11][12][13][14]. Systems for cell barcoding include direct introduction of barcodes via lentiviral infection, CRISPR-based barcoding, or a combination of barcoding with fluorescent markers [9][10][11][14][15][16][17]. A very important advantage of these systems is high sensitivity and the ability to simultaneously monitor the fate of thousands and even millions of cells [5,6,18].…”

SunCatcher: Clonal Barcoding with qPCR Detection Enables Live Cell Functional Analyses to Study Cancer Heterogeneity, Progression, and Metastasis

“…While such high-complexity barcode libraries enable longitudinal quanti cation of clonal diversity in various experimental settings, they do not enable characterization, functional analyses, or manipulation of the cellular populations of interest. More recently, a number of elegant and sophisticated methods have been developed to trace as well as study certain select barcoded cells [30][31][32][33][34] ; however, some of those methods are limited in the numbers of barcoded cells that can be selected for study, and the techniques are not widely accessible.…”