With the ability to easily identify and isolate single cells from a heterogeneous phenotypic population, a cell sorter is a useful tool for laboratories studying and applying the CRISPR technology as a tool for editing genes and creating single cell libraries.

CRISPR genome editing tools can be used to remove, change or insert DNA sequences into genes. The resulting mutations are maintained in the genome as cells proliferate –in contrast to transient transfection methods.

Edits to genes are accomplished by directing the Cas9 nuclease to cleave DNA at a specific sequence and a guide RNA (gRNA) into the cell through transfection (or electroporation). For the edit to be completed the cell must be successfully transfected and the DNA cleaved by the Cas9 (referred to as cleavage efficiency).

Many factors can impact cleavage efficiency including the efficiency of the transfection. A commonly used method to identify cells that have been successfully transfected is to include a plasmid encoding for a fluorescent protein such as eGFP in the transfection along with Cas9 and gRNA.

Cells can then be sorted based on the expression of the fluorescent protein. The SH800 sorter, lets researchers deposit target cells into 96- or 384 multi-well devices and indexing software records the X and Y well position of each cell. The software can also correlate the fluorescence and scatter phenotype of sorted cells for meta-data analysis.

The Sony SH800 has been used by several prominent research laboratories to optimize CRISPR expressing cells and to create CRISPR variants with improved function. For more information download this Application Data that demonstrates how the Sony SH800S can be used for single cell sorting of CRISPR/CAS 9 expressing cells.

Other laboratories have used the SH800 for routine sorting of CRISPR modified cells. Learn more about sorting CRISPR modified cells in the publications below:

 

 


References

Oakes, Benjamin L., et al. "Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch." Nature biotechnology (2016). PubMed

Slush, Valentin M., et al. "Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line." Scientific reports 5 (2015). PubMed

Goto, Teppei, et al. "Hypomorphic phenotype of Foxn1 gene-modified rats by CRISPR/Cas9 system." Transgenic research (2016): 1-12. PubMed

Oakes, B, et al. "Protein engineering of Cas9 for enhanced function." Methods in enzymology 546 (2014): 491. PubMed

Schrage, Ramona, et al. "The experimental power of FR900359 to study Gq-regulated biological processes." Nature communications 6 (2015). PubMed

Hayashi, Masayasu, et al. "Chd5 Regulates MuERV‐L/MERVL Expression in Mouse Embryonic Stem Cells Via H3K27me3 Modification and Histone H3. 1/H3. 2." Journal of Cellular Biochemistry (2015). PubMed