Research Article

Developmental barcoding of whole mouse via homing CRISPR

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Science  31 Aug 2018:
Vol. 361, Issue 6405, eaat9804
DOI: 10.1126/science.aat9804

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Lineage tracing in mouse using CRISPR

A homing guide RNA (hgRNA) that directs CRISPR-Cas9 to its own DNA locus can diversify its sequence and act as an expressed genetic barcode. Kalhor et al. engineered a mouse line carrying 60 independent loci of hgRNAs, thus generating a large number of unique barcodes in various embryonic and extraembryonic tissues in fully developed mice. This method demonstrates lineage tracing from the very first branches of the development tree up to organogenesis events and was used to elucidate embryonic brain patterning.

Science, this issue p. eaat9804

Structured Abstract

INTRODUCTION

The remarkable development of a single cell, the zygote, into the full organism occurs through a complex series of division and differentiation events that resemble a tree, with the zygote at the base branching through lineages that end in the terminal cell types at the top. Characterizing this tree of development has long been a subject of interest, and the combination of modern genome engineering and sequencing technologies promises a powerful strategy in its service: in vivo barcoding. For in vivo barcoding, heritable random mutations are induced to accumulate during development and sequenced post hoc to reconstruct the lineage tree. Demonstrations thus far have largely focused on lower vertebrates and have used a barcoding element with a constrained window of activity for clonal tracing of individual cells or cell types. Implementation in mammalian model systems, such as the mouse, incurs unique challenges that require major enhancements.

RATIONALE

To address the complexity of mammalian development, we reasoned that multiple independent in vivo barcoding elements could be deployed in parallel to exponentially expand their recording power. Independence requires both an absence of cross-talk between the elements and an absence of interference between their mutation outcomes. A system with the potential to deliver on these requirements is homing CRISPR, a modified version of canonical CRISPR wherein the homing guide RNA (hgRNA) combines with CRISPR-Cas9 nuclease for repeated targeting of its own locus, leading to diverse mutational outcomes. Therefore, in mouse embryonic stem cells, we scattered multiple hgRNA loci with distinct spacers in the genome to serve as barcoding elements. With this arrangement, each hgRNA acts independently as a result of its unique spacer sequence, and undesirable deletion events between multiple adjacent cut sites are less likely. Using these cells, we generated a chimeric mouse with 60 hgRNAs as the founder of the MARC1 (Mouse for Actively Recording Cells 1) line that enables barcoding and recording of cell lineages.

RESULTS

In the absence of Cas9, hgRNAs are stable and dormant; to initiate barcoding, we crossed MARC1 mice with Cas9 knock-in mice. In the resulting offspring, hgRNAs were activated, creating diverse mutations such that an estimated 1023 distinct barcode combinations can be generated with only 10 hgRNAs. Furthermore, hgRNAs showed a range of activity profiles, with some mutating soon after conception while others exhibited lower activity through most of the gestation period. This range resulted in sustained barcoding throughout gestation and recording of developmental lineages: Each cell inherits a set of unique mutations that are passed on to its daughter cells, where further unique mutations can be added. Consequently, at any stage in such developmentally barcoded mice, closely related cells have a more similar mutation profile, or barcode, than the more distant ones. These recordings remain embedded in the genomes of the cells and can be extracted by sequencing.

We used these recordings to carry out bottom-up reconstruction of the mouse lineage tree, starting with the first branches that emerged after the zygote, and observed robust reconstruction of the correct tree. We also investigated axis development in the brain by sequencing barcodes from the left and right side of the forebrain, midbrain, and hindbrain regions. We found that barcodes from the left and right sides of the same region were more closely related than those from different regions; this result suggests that in the precursor of the brain, commitment to the anterior-posterior axis is established prior to the lateral axis.

CONCLUSION

This system provides an enabling and versatile platform for in vivo barcoding and lineage tracing in a mammalian model system. It can straightforwardly create developmentally barcoded mice in which lineage information is prerecorded in cell genomes. Combining multiple independently acting molecular recording devices greatly enhances their capacity and allows for reliable information recovery and reconstruction of deep lineage trees.

Developmental barcoding and lineage reconstruction in mice.

Crossing the MARC1 mouse line, which carries multiple hgRNAs, with a CRISPR-Cas9 mouse line results in developmentally barcoded offspring that record lineages in their cells. These recordings were extracted and used to reconstruct lineage trees. A combination of the trees extracted from different developmentally barcoded mice is shown. ICM, inner cell mass; E0, embryonic day 0.

Abstract

In vivo barcoding using nuclease-induced mutations is a powerful approach for recording biological information, including developmental lineages; however, its application in mammalian systems has been limited. We present in vivo barcoding in the mouse with multiple homing guide RNAs that each generate hundreds of mutant alleles and combine to produce an exponential diversity of barcodes. Activation upon conception and continued mutagenesis through gestation resulted in developmentally barcoded mice wherein information is recorded in lineage-specific mutations. We used these recordings for reliable post hoc reconstruction of the earliest lineages and investigation of axis development in the brain. Our results provide an enabling and versatile platform for in vivo barcoding and lineage tracing in a mammalian model system.

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