title: Rapidly evolving homing CRISPR barcodes.
abstract: We present an approach for engineering evolving DNA barcodes in living cells. A homing guide RNA (hgRNA) scaffold directs the Cas9-hgRNA complex to the DNA locus of the hgRNA itself. We show that this homing CRISPR-Cas9 system acts as an expressed genetic barcode that diversifies its sequence and that the rate of diversification can be controlled in cultured cells. We further evaluate these barcodes in cell populations and show that they can be used to record lineage history and that the barcode RNA can be amplified in situ, a prerequisite for in situ sequencing. This integrated approach will have wide-ranging applications, such as in deep lineage tracing, cellular barcoding, molecular recording, dissecting cancer biology, and connectome mapping.
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Reza
authors.identifier: 0000-0002-5558-7545
authors.initials: R
authors.lastname: Kalhor
authors.affiliation: Department of Bioengineering, University of California San Diego, La Jolla, California, USA.
authors.forename: Prashant
authors.initials: P
authors.lastname: Mali
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.|Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA.
authors.forename: George M
authors.initials: GM
authors.lastname: Church
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D049452:Green Fluorescent Proteins; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases