A thermostable Cas9 with increased lifetime in human plasma

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.


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This temperature restriction is particularly limiting for genome editing in obligate 49 thermophiles 11 . Recent efforts using SpyCas9 to edit a facultative thermophile have 50 been possible by reducing the temperature within the organism 12 . While effective, this 51 approach is not feasible for obligate thermophiles, and requires additional steps for 52 moderate thermophiles. This is especially important for metabolic engineering for which 53 thermophilic bacteria present enticing hosts for chemical synthesis due to decreased risk 54 of contamination, continuous recovery of volatile products and the ability to conduct 55 reactions that are thermodynamically unfavorable in mesophilic hosts 13 . Developing a 56 thermostable Cas9 system will enable facile genome editing in thermophilic organisms 57 using technology that is currently restricted to mesophiles.

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CRISPR-Cas9 has also emerged as a potential treatment for genetic diseases 14 .

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A promising method for the delivery of Cas9 into patients or organisms is the injection of 60 preassembled Cas9 ribonucleoprotein complexes (RNP) into the target tissue or 61 bloodstream 15 . One major challenge to this approach is that Cas9 must be stable 62 4 enough to survive degradation by proteases and RNases in the blood or target tissue for 63 efficient delivery. Limited protein lifetime will require delivery of higher doses of Cas9 into 64 the patient or result in poor editing efficacy. In contrast, delivering a Cas9 with improved 65 stability could greatly enhance genome-editing efficiency in vivo.

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To address these challenges, we tested the thermostable Cas9 protein from 67 Geobacillus stearothermophilus (GeoCas9). We find that GeoCas9 maintains activity 68 over a wide temperature range. By harnessing the natural sequence variation of 69 GeoCas9 from closely related species, we engineered a protospacer adjacent motif 70 (PAM)-binding variant that recognizes additional PAM sequences and thereby doubles 71 the number of targets accessible to this system. We also engineered a highly efficient Bacillus stearothermophilus) 18 stood out because it was full-length and its sequence is 87 shorter than that of the average Cas9. Most importantly, this candidate is found in an = 88 5 organism that can grow at a reported temperature range of 30°C-75°C (optimal at 89 55°C

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We purified GeoCas9 and performed initial thermostability tests using differential 104 scanning calorimetry (DSC), which showed that in the absence of RNA or DNA,

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In addition to the CRISPR loci found in G. st. strains, we also found a type II 123 CRISPR locus in Geobacillus LC300 containing a Cas9 with ~97% amino acid identity to tested cleavage activity on targets containing various PAM sequences (Fig. 2b). We 134 found that, as predicted by protospacer sequences, the hybrid Cas9 preferred a GMAA 135 PAM rather than the CRAA PAM utilized by GeoCas9. Moreover, G. LC300 appears to 136 be more specific for its optimal PAM, which may result in lower off-target cleavage for

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Often thermostability comes at the cost of reduced activity at lower 202 temperatures 33 . However, the wide range of natural growth temperatures for G. st.

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suggested that GeoCas9 might maintain activity at mesophilic temperatures. To examine 204 this hypothesis, we measured the cleavage rate of SpyCas9 and GeoCas9 at various 205 temperatures (Fig. 5b). SpyCas9 DNA cleavage rates increased between 20-35°C, 206 reaching maximum levels from 35-45°C. Above these temperatures, SpyCas9 activity 207 dropped sharply to undetectable levels, as predicted by thermostability measurements.

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In contrast, GeoCas9 activity increased to a maximum measured value at 50°C and 209 maintained maximum detectable activity up to 70°C, dropping to low levels at 75°C.

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These results make GeoCas9 a valuable candidate for editing obligate thermophilic 211 organisms and for biochemical cleavage applications requiring Cas9 to operate at 212 elevated temperatures.

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It was shown previously that thermostable proteins have longer lifetime in 214 blood 34 . To test if this is the case for GeoCas9, we incubated SpyCas9 and GeoCas9 in 215 diluted human plasma at 37°C for 8 hrs and measured the amount of Cas9 activity 216 remaining (Fig. 5c). Although SpyCas9 maintained activity when incubated in reaction 217 10 buffer at 37°C, its activity was abolished even at the lowest concentration of plasma. In 218 contrast, GeoCas9 maintained robust activity after incubation with human plasma, 219 making it a promising candidate for in vivo RNP delivery.

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Our results establish GeoCas9 as a thermostable Cas9 homolog and expand the 223 temperatures at which Cas9 can be used. We anticipate that the development of 224 GeoCas9 will enhance the utility of CRISPR-Cas9 technology at both mesophilic and 225 thermophilic temperatures. The ability of Cas9 to function reliably in a wide range of 226 species has been key to its rapid adoption as a technology, but the previously developed

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We were interested to note that GeoCas9 and SpyCas9 induced similar levels of 235 indels in HEK293T cells as SpyCas9 when delivered as an RNPs (Fig. 4b-d), despite 236 GeoCas9's lower DNA cleavage rate at 37°C (Fig. 5b). We conclude that biochemical 237 cleavage rates may not reflect limiting step of target search in a human cell. It may be 238 that GeoCas9 can persist longer in cells, which raises its effective concentration over 239 time and compensates for its slower cleavage rate. Moreover, in applications requiring 240 delivery of Cas9 into the bloodstream, the benefit of improved stability by GeoCas9 may 241 become even more apparent.

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The development of GeoCas9 hinged upon utilizing the naturally occurring 243 diversity of CRISPR systems. The sheer abundance and diversity of Cas9 makes it 244 advantageous over newer type V systems, such as Cpf1, for developing specialized 245 genome editing tools. It has previously been suggested that type II CRISPR systems are 246 only found in mesophilic bacteria, and that protein engineering would be required to

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For thermostability measurements (Fig. 4a), 100nM Cas9 was complexed with 407 150nM sgRNA in 1x Reaction Buffer for 5min at 37°C. 100nM of a PCR product 408 containing the targeted sequence was cleaved using dilutions of the estimated 100nM 409 RNP complex to accurately determine a 1:1 ratio of Cas9 to target. Next, samples were 410 challenged at the indicated temperature (40°C-75°C) for 10min and then returned to 411 37°C. 100nM PCR product containing the targeted sequence was added to the reaction 412 and it was allowed to react for 30min at 37°C. The reaction was quenched with 6×