Purification of Cas9 with SRT-10 SEC 300

***Special callout as the chromatographer who performed this work, William Selleck , is available for consulting work!

Explain Like I'm Five | ELI5

To my friends and family not in the biopharma industry:

• The human genome is analogous to an extensive instruction manual, with each page containing directives for the functionality of various bodily components.
• Occasionally, errors occur within these pages, resulting in illness. Genome editing is a methodology employed by scientists to rectify these errors.?
• Utilizing Cas9 and guide-RNA, researchers have developed a novel approach to amend these inaccuracies via cationic lipid delivery.
• This technique has demonstrated efficacy in correcting errors while minimizing off-target activity, rendering this research significant.

Biophysical Characterization and Direct Delivery of S. pyogenes Cas9 Ribonucleoprotein Complexes

Several groups have demonstrated efficient genome editing in various mammalian cells by cationic lipid mediated delivery of purified Cas9 protein complexed with guide-RNA (gRNA). This direct delivery of the Cas9 ribonucleoprotein (RNP) complex allows for efficient gene-editing while minimizing off-target activity.

?? The Cas9 complex with gRNA has been well characterized structurally and biophysically.

??? Thermal melt curves are used to detect the binding and stability of complexes.

?? Multiple sgRNAs with differing lengths and base composition were complexed with Cas9.

?? These biophysically characterized complexes were then transfected into 293T cells and the efficiency of indel generated was measured.

?? Subtle differences in the sgRNA length and base composition affect the binding and formation of RNP complex.

?? Correlating binding affinity with efficiency of genome editing informs the design of an optimal composition of RNPs for cationic lipid mediated direct delivery.

Notes:

Size Exclusion Chromatography Column

• Sepax Technologies, Inc.?SRT-10 SEC-300, 10um, 300 A 21.2 x 300 mm
• Part Number:?225300-21230

Citation

Selleck, William, et al. "Biophysical Characterization and Direct Delivery of S. pyogenes Cas9 Ribonucleoprotein Complexes." Editas Medicine, Apr 27 (2015).

https://www.editasmedicine.com/wp-content/uploads/2017/09/ASGCT-poster_2015_Will.pdf

Authors

William Selleck , Hari Jayaram , G. Grant Welstead , David Bumcrot , Anne Bothmer

Author Affiliations @ Time of Publication

Editas Medicine , Cambridge, MA, 02142, USA