Purifying Cas Proteins and Complexes: Part II

March 10, 2020

This blog post is the second in a series that spotlights proteins that have been purified by the CL7/Im7 system. This is the second of two installments detailing Cas9 purification.

Cas9 requires high purity in order to achieve high activity as a part of the CRISPR system for genetic modification. Researchers from Hubei University in Wuhan, China used TriAltus’ CL7/Im7 system successfully to purify Cas protein and Cas RNP complexes. Due to a growing demand for pure Cas proteins and an effort to improve their production, TriAltus conducted its own purification runs of Cas9 with success.

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TriAltus’ Cas9 purification

After the Hubei group’s successful purification of Cas9 RNPs, TriAltus ran its own tests of Cas9 purification. In order to prove the efficacy of CL7 in purifying a key protein, two identical runs were conducted: one using His8 and SUMO tags and one using CL7 and MBP tags. The two workflows are illustrated in Figure 1.

Figure 1. Workflow for His-tag and CL7 tag purifications of Cas9 protein.

Loading/lysis buffer was consistent between the two runs at 2M NaCl, and both used 5 ml of chromatography beads. Cas9/His8 resulted in only about 60% purity, with the contaminants being leftover proteins from E. coli. In contrast, Cas9/CL7 achieved near-perfect purity of 99%. Cas9/His8 only achieved 95% purity after the 4th step (Figure 2A). When the Im7 column run was scaled up to 100g of cells and the optional second step was used to remove trace amounts of PSC protease, the product was 99% pure (Figure 2B).

Figure 2. Cas9 was expressed in E. coli and lysates were purified using the schemes outlined in the methods section. Fractions were analyzed on 4-15% SDS-PAGE and Coomassie-stained. A) Eluate obtained from a small-scale (5 mL) His-Trap column compared with eluate from a small-scale (5 mL) Im7 column. The final product after 4 steps is shown in the final lane for comparison to the final product after 1 step in the penultimate lane. B) Cas9WT from a commercial source (CO) compared with the small-scale Cas9WT from Im7 column, the large-scale Cas9WT from the Im7 column, and the large-scale high-fidelity (HF) mutant Cas9 followed by a GST “polishing” step to remove PSC P. 15 µg of protein was loaded per lane.

Proven Cas9 activity

Cas9 obtained from a commercial source (CO) or CL7/Im7-purified Cas9 (Im7) was complexed with equimolar (+), or without (-), an annealed sgRNA (Figure 3). Complexes were incubated at different concentrations with a 1.1 kb target DNA fragment for 15 minutes at 37°C. The sgRNA recognizes a sequence in the middle of the target DNA, resulting in ~550 bp fragments upon cleavage. CL7-purified Cas9 showed comparable activity to commercial Cas9.

Figure 3. CL7/Im7-purified-Cas9’s cleavage activity compared to commercially obtained Cas9. Reactions were resolved on a 1.2% agarose/TAE gel which was then stained with SYBR GOLD.

CL7-purified Cas9 also showed the same activity in cells as the His8/four-step-purified Cas9. Human sickle cell iPSCs (induced pluripotent stem cells) were electroporated with Lonza nucleofector 2b and varying concentrations of RNP using Cas9WT from either the 4-step purification (His8) or 1-step purification protocols (CL7). RNPs made with Cas9 purified using the two protocols were tested using a sgRNA designed to correct a 1-bp error in hemoglobin that causes sickle cell disease. Modification rates were evaluated by digital PCR. The proteins resulted in the same viability and modification rates, indicating equivalent efficiency in cells. These tests were performed by Tim Townes and Lei Ding of the University of Alabama Birmingham (UAB).

Conclusion

Cas9 will continue to be an essential component in the refinement and development of CRISPR technology. As more scientists turn their attention to CRISPR and Cas9, it will be important that they use the highest quality Cas9 products to generate optimal results. The CL7/Im7 tag system has emerged as a strong candidate for producing high-quality Cas9 and will continue to play a role in its future use.

Read Part I of this blog post about Hubei University researchers’ purification of Cas9 RNPs.