Purifying Cas Proteins and Complexes: Part I
This blog post is the first in a series that spotlights proteins that have been purified by the CL7/Im7 system. This is the first of two installments detailing Cas9 purification.
In the past several years, CRISPR has emerged as a powerful system for targeted gene modification. The system requires only two primary components: a guide RNA (gRNA or sgRNA) sequence and a Cas9 enzyme. Cas9 is essential for initiating a double-stranded break in the gene sequence at the site to which the gRNA guides it. In order to achieve high-fidelity results, the Cas9 protein must be pure and active.
Why use ultra-pure Cas9?
Purity and activity are largely determined by the method of purification that is used to isolate the Cas9 protein. Incumbent methods require multiple purification steps that can include combinations of metal affinity enrichment, cation exchange chromatography, size exclusion chromatography, and tag-based affinity chromatography. Depending on multiple chromatography steps for purification is not only time consuming, but also results in product losses at each phase and therefore, a lower final yield. Recently, researchers at Hubei University (Wuhan, China) used the CL7/Im7 system to purify Cas9 ribonucleoproteins (RNPs) in one step, saving time and maintaining high activity. This blog post describes the results of Hubei’s Cas purifications.
Cas RNP purification
In May of 2019, a research group from Hubei University in China published a paper validating TriAltus’s claims to success with the CL7/Im7 system. Their need for highly pure Cas9 protein stemmed from their study of the co-expression of Cas9 and sgRNAs in E. coli.
One of the major issues with CRISPR is its method of delivery into cells. A new and promising method is the direct assembly of ribonucleoprotein (RNP) complexes with Cas proteins in E. coli. Traditional approaches mix separately constructed sgRNAs and Cas proteins in vitro, which can have low success rates due to potential degradation of the sgRNA by RNAses in solution. Another method is to introduce a DNA expression cassette with the Cas and sgRNA genes so that the cell makes both elements and they assemble. However, this results in more off-target effects because the Cas9 DNA has more longevity in the cell.
Alternatively, the Hubei group developed a method of direct co-expression of Cas9 and sgRNA in E. coli before purification of the RNP. Co-expression results in spontaneous self-assembly of the components into the full Cas9 RNP complex. Precomplexed RNPs are reported to be more stable than ones mixed in vitro. The success of this method is contingent upon the purity of their Cas9 RNP complex.
The CL7 tag was inserted at the N-terminus of Cas9, and Cas9 RNP was purified with a Ni-NTA column and an Im7 column. Using CL7/Im7, they reported a yield of ~40 mg/L cells, a 4x increase compared to traditional methods. Purity measured by densitometry also increased from ~58% to 89% (Figure 1). They repeated the test with Cas12a RNP and saw a similar jump in purity from 61% to 87% with a 30 mg/L yield, a 3x increase (Figure 1).
Proven Cas RNP activity
Furthermore, the Hubei researchers verified the enzymatic activity of the Cas RNPs. In 30 minutes at 37° C, 300 ng of plasmids were fully cleaved by 200 ng of Cas RNPs- Cas9, CL7-Cas9, and Cas12a RNP. Significantly, the RNP unit showed no difference in cleavage ability with the presence of the CL7 tag on the CL7-Cas9 RNP. This suggests that the CL7 tag is non-interactive with DNA.
Additionally, homology-directed repair (HDR) efficiency was 1.8 times higher (19%) when the CL7/Im7-purified Cas9/RNP unit was transfected into BFP-HEK293 cells than when the RNPs were purified with traditional methods (11%).
This data supports the claims that the CL7/Im7 system can purify complex proteins at very high purity levels while achieving similar or higher activity.