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Rapidly evolving homing CRISPR barcodes


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.

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Figure 1: Schematic representation of a lineage-tracing approach in multicellular eukaryotes using genome engineering and single cell DNA sequencing.
Figure 2: Comparison between standard and homing CRISPR–Cas9 systems.
Figure 3: Performance of various hgRNAs.
Figure 4: Lineage tracing in cultured cell populations.

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The authors would like to acknowledge W.L. Chew, J. Aach, S. Byrne, E. Daugharthy, T. Ferrante, J.H. Lee, M. Moosburner, I. Peikon, H. Lee, A. Ng, J. Fernandez Juarez, A. Marblestone, A. Chavez, Y. Mayshar, J. Scheiman, K. Kalhor, T. Wu, J. Shendure, and T. Lu for helpful comments or discussions and the Biopolymers Facility at HMS for technical assistance. This work has been supported by funding from NIH grants MH103910 and HG005550 (G.M.C.) and the Intelligence Advanced Research Projects Activity (IARPA) via Department of Interior/Interior Business Center (DoI/IBC) contract number D16PC00008 (G.M.C.), and by UCSD new faculty startup funds (P.M.).

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Authors and Affiliations



R.K., P.M., and G.M.C. conceived the study. R.K. and P.M. carried out the experiments. R.K. analyzed the data. R.K. and P.M. wrote the manuscript.

Corresponding authors

Correspondence to Prashant Mali or George M Church.

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Competing interests

R.K., P.M. and G.M.C. have filed provisional patent applications based on this study.

Integrated supplementary information

Supplementary Figure 1 Comparing canonical and homing guide RNAs.

(a) Primary sequences and the predicted secondary structures of canonical and homing gRNAs. Positions that were mutated to derive the hgRNA are underlined. Orange bases mark the newly created PAM in hgRNA. Grey boxes mark all the positions where hgRNA is mutated compared to canonical sgRNA. (b) Results of a HR-based assay to evaluate to functionality of hgRNAs. A genomically integrated GFP coding sequence is disrupted by the insertion of a stop codon and a 68-bp genomic fragment from the AAVS1 locus. Restoration of the GFP sequence by HR with an appropriate donor sequence results in GFP+ cells that can be quantified by FACS. AAVS1 locus contains a site known as T1 which matches the spacer sequence of hgRNA-A21. Bar graph on top depicts HR efficiencies induced by wild type and homing T1 guide RNAs, as measured by FACS. Representative FACS plots of the targeted cells are depicted below. Data are shown as means ± s.e.m (n=4). (c) Sequencing results showing above-background accumulation of mutations in hgRNA locus upon Cas9 expression. Cas9 expression is induced in cells with a lentivirally integrated hgRNA-A21. DNA samples harvested before (t=0 days) and at various points after induction are characterized by high-throughput sequencing to quantify mutations in the DNA locus that encodes hgRNA-A21. Any mismatch, deletion, or insertions compared to the original locus is considered a mutation. The high initial fraction of mutants is due to sequencing error (materials and methods) as the steady level of mutations in the non-induced sample suggests there is no significant Cas9 expression leakage in the course of our experimental times. (d) Comparison of viability between cells containing hg and sgRNAs. Cells containing an sgRNA were mixed with cells containing an hgRNA with the same spacer sequence on day 0. Both cells also included the target site for the sg and hgRNA. Cas9 expression was induced and samples were taken at various points after induction up to 20 days. Sequencing was then used to identify the fraction of cell at each point with each gRNA as represented by the fraction of reads that match each kind of scaffold (Methods). The results show a similar viability for cells carrying sgRNAs and hgRNAs.

Supplementary Figure 2 Longer variants of homing gRNAs.

Design of four longer variants of hgRNA-A21 is shown. Stuffer sequences of 5, 30, 55, or 80 base pairs were added upstream of a spacer very similar to the A21 spacer to obtain the four increasingly lengthy A’ variants. (B,C,D) Cas9 is induced in cell lines with the hgRNAs from panel A integrated lentivirally. All samples were harvested in the end; however, induction was initiated at 1, 2, 3, or 4 weeks before harvest with one sample was not being induced (0 weeks of induction). DNA was extracted from all samples and characterized by high-throughput sequencing to quantify mutations and functionality of hgRNA loci. Any mismatch, deletion, or insertion compared to the original locus is considered a mutation. The high pre-induction fraction of mutants is likely due to a combination of sequencing error (materials and methods) and background Cas9 expression leakage which becomes a significant factor during the 4-week course of the experiment. The drop in total diversity of hgRNA-A21 after the second week of induction is likely due to loss of diversity due to the bottle-necking effect of passaging the cells.

Supplementary Figure 3 Target-specific in situ detection of gRNA molecules expressed by RNA polymerase III.

(a) A schematic of our in situ amplification and detection assay based on FISSEQ. (b) Results of target-specific in situ amplification and detection for two different hgRNA constructs and a negative control. The schematic on top shows the position of the reverse-transcription (RT) primer in each design. The bottom panels show a representative field of view from each of two biological replicates. Amplicons are labeled with Cy5 (orange) and nuclei are labeled with DAPI (blue).

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Supplementary Figures 1–3, Supplementary Tables 1 and 2, and Supplementary Note (PDF 1878 kb)

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Kalhor, R., Mali, P. & Church, G. Rapidly evolving homing CRISPR barcodes. Nat Methods 14, 195–200 (2017).

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