A thermostable Cas9 with increased lifetime in human plasma

Abstract

CRISPR-Cas9 is a powerful technology that has enabled genome editing in a wide range of species. However, the currently developed Cas9 homologs all originate from mesophilic bacteria, making them susceptible to degradation and unsuitable for applications requiring cleavage at elevated temperatures. Here, we show that the Cas9 protein from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) catalyzes RNA-guided DNA cleavage at elevated temperatures. GeoCas9 is active at temperatures up to 70 °C, compared to 45 °C for Streptococcus pyogenes Cas9 (SpyCas9), which expands the temperature range for CRISPR-Cas9 applications. We also found that GeoCas9 is an effective tool for editing mammalian genomes when delivered as a ribonucleoprotein (RNP) complex. Together with an increased lifetime in human plasma, the thermostable GeoCas9 provides the foundation for improved RNP delivery in vivo and expands the temperature range of CRISPR-Cas9.

Fig. 1

GeoCas9 is a thermostable Cas9 homolog. a Phylogeny of Cas9 proteins used for genome editing with their length (amino acids) and the maximum temperatures that supports growth of the host indicated to the right. b Homology model of GeoCas9 generated using Phyre 2 with the DNA from PDB 5CZZ docked in. c Schematic illustration of the domains of Spy Cas9 (blue) and GeoCas9 (orange) with active site residues indicated below with asterisks. d Representative traces for differential scanning calorimetry (DSC) of GeoCas9 and SpyCas9. e Denaturation temperature of various Cas9 proteins as measured by DSC, mean ± S.D. is shown. Nme Neisseria meningitides, Geo Geobacillus stearothermophilus, Geo LC300 Geobacillus LC300, Spy Streptococcus pyogenes, Sau Streptococcus aureus, Fno Francisella novicida, Sth (3) Streptococcus thermophilus CRISPR III, T _d denaturation temperature

Fig. 2

PAM identification and engineering of GeoCas9. a WebLogo for sequences found at the 3′ end of protospacer targets identified with CRISPRTarget for Geobacillus stearothermophilus (left) and Geobacillus LC300 (right). b Cleavage assays conducted with the two homologs of GeoCas9. Substrates with various PAM sequences were ³²P-labeled and mean ± S.D. is shown. c Mapping of mutated residues (orange spheres) between G. st. and G. LC300 onto the homology model of GeoCas9 showing high density in the PAM-interacting domain near the PAM region of the target DNA. d Alignment of the Cas9 proteins from G. st. and G. LC300 with the domain boundaries shown above. Solid colors represent identical residues and gray lines indicate residues that are mutated between the two Cas9 homologs

Fig. 3

Small RNA-seq and sgRNA engineering for GeoCas9. a Small RNA sequenced from G. stearothermophilus mapped to the CRISPR locus. Inset shows enlargement of the region corresponding to the tracrRNA and the most highly transcribed repeat and spacer sequence. b Distribution of the length of the spacer sequences extracted from the small RNA sequencing results. c Length optimization of the tracrRNA and crRNA for GeoCas9 and the optimal guide RNA design (right). The length of the tracrRNA, crRNA:tracrRNA duplex and spacer was optimized sequentially by transcribing variations of the sgRNA and testing their ability to guide GeoCas9-mediated cleavage of a radiolabeled substrate. For spacer length, two different targets were used (Target 1 and Target 2). The mean k _cleave ± S.D. is shown and experiments were conducted in triplicate

Fig. 4

Genome-editing activity of GeoCas9 in mammalian cells. a EGFP disruption in HEK293T cells by GeoCas9. HEK293FT cells expressing a destabilized GFP were transfected with GeoCas9 RNP preassembled with a targeting or non-targeting guide RNA. Cells were analyzed by flow cytometry and targets adjacent to the CRAA PAM resulted in efficient GFP disruption. b T7E1 analysis of indels produced at the AAVS1 locus when the guide length was varied from 21 to 22 nt. The Cas9 used is indicated above each lane and the length of the spacer is shown below. c T7E1 analysis of indels produced using a titration of GeoCas9 and SpyCas9 RNP targeting the DNMT1 locus in HEK293T cells. The Cas9 used is indicated above each lane and the amount of RNP delivered to each well of a 96-well plate is indicated below. NT non-targeting

Fig. 5

Thermostability of GeoCas9 and longevity in human plasma. a Activity of SpyCas9 and GeoCas9 after incubation at the indicated temperature. After challenging at the higher temperature, reactions were conducted at 37 °C using a 1:1 ratio of substrate to RNP. b Cleavage rate of SpyCas9 and GeoCas9 RNPs at various temperatures. Maximum detection limit is shown by the dashed line at k _cleave = 5, indicating that the reaction completed in ≤30 s. c Lifetime of SpyCas9 after incubation in 50% mouse plasma. Incubation was done at 37 °C for the specified amount of time after which DNA substrate was introduced at an equimolar ratio of substrate to RNP. d Effect of incubating GeoCas9 and SpyCas9 in human plasma. After incubation in varying concentrations of human plasma for 8 h at 37 °C, the reaction was carried out with 1:1 ratio of DNA substrate to RNP