In Vitro Revival: Primordial Germ Cell Isolation, Culturing, and CRISPR/Cas9 Genome Editing

To produce genome-edited birds the most efficient method begins with editing genomes in cell cultures. For birds, the only cells that can be used are Primordial Germ Cells (PGCs) – a kind of embryonic stem cell that becomes sperm and eggs in adult birds.

In a developing embryo, PGCs can be isolated from the germinal crescent, a region in the early-stage embryo where those particular cells form, or from the gonads at a later stage in embryonic development. By studying Rock Pigeon embryonic development, our partner Crystal Bioscience has developed the protocols for isolating PGCs.

Once isolated, these cells can be grown in a liquid medium of nutrients called the culture. Cultured cells will live and grow in the lab, allowing us to make many gene changes without needing to hatch birds every time we make edits to the genome. The right culture conditions for different bird species requires experimentation to develop the correct formula. Developing PGC cultures for pigeons is essential to accomplishing de-extinction.

Once PGCs are in culture, we can use CRISPR/Cas9 technology to edit the genome. The CRISPR/Cas9 system is a “cut and paste” biotechnology – the CRISPR portion works to direct the Cas9 enzyme to a specific site in the genome, where the tool binds and cuts. Once cut, the cell’s own DNA repair mechanisms set to work to mend the break in the DNA molecule – this is an opportunity to introduce alternate DNA codes at the site of the cut.

[A] We will design CRISPR sequences to target specific regions of the genome containing the genes responsible for differences between Band-tailed Pigeons and Passenger Pigeons. Once our custom CRISPR/Cas9 complexes are injected inside a cell, they will bind to the Band-tailed Pigeon genome at those target sites and cut the DNA.

[B] The synthesized Passenger Pigeon DNA from Phase 1.3 is introduced to the cell with the CRISPR/Cas9. After the DNA is cut, the synthesized DNA is integrated into the genome by the cell’s own DNA repair mechanism – homologous recombination. The ends of the synthesized Passenger Pigeon DNA code will match the DNA at the cut sites, allowing the cell to piece together the two strands of codes by binding the matching ends.

[C] Now that the Band-tailed Pigeon DNA has been removed and overwritten with Passenger Pigeon DNA, the process of “allele replacement” is complete. This means the cultured PGCs are now slightly Passenger Pigeon. By repeating this process, we will eventually develop PGCs that contain newly created Passenger Pigeon genomes resembling a sort of hybrid DNA code between modern Band-tailed Pigeons and extinct Passenger Pigeons.

The Goal

The goal is a hybrid genome that produces a bird that looks and behaves like an extinct Passenger Pigeon and that the genetic legacy of the extinct species.