GESTALT

[1] GESTALT involves introducing a small DNA barcode that contains regularly spaced CRISPR/Cas9 target sites into the genomes of progenitor cells.

These methods use recombinases, the two main ones being the Cre-loxP and Flp-firt systems, which can delete segments of DNA flanked by the loxP and frt sites, respectively.

[5][6] In this method, a transgenic model is created that can express Cre recombinase and has a reporter gene with an upstream stop cassette flanked by loxP sites.

[8] Although powerful, it requires significant optimization to facilitate single cell lineage tracing and is low throughput.

[9][10] Early approaches leveraged naturally occurring somatic mutations to identify cell lineage relationships.

[11] GESTALT takes advantage of the CRISPR-Cas9 system, which allows for the targeting of double stranded breaks in DNA to highly specific sites adjacent to PAM motifs based on the sequence of the sgRNA.

[12] Non-homolgous end joining is the more active of the two repair pathways, resulting in indels occurring at the targeted site.

The first method transduces progenitor cells with a lentivirus construct containing the barcode array inserted into the 3'-UTR of EGFP.

[1] Following delivery of the CRISPR-Cas9 reagents, time is allowed for barcode editing and further development to occur, resulting in the expansion of the labeled populations and the unique marking of their progeny.

Unique molecular identifiers are used to correct for PCR bias and each UMI-barcode combo is therefore from a single cell.

GESTALT was initially developed to examine the contributions of embryonic progenitors to the adult organ systems of zebrafish.