A thermostable Cas9 with increased lifetime in human plasma

Harrington, Lucas B.; Páez-Espino, David; Chen, Janice S.; Ma, Enbo; Staahl, Brett T.; Kyrpides, Nikos; Doudna, Jennifer A.

doi:10.1101/138867

Cited by 36 publications

(55 citation statements)

References 43 publications

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“…To test this idea, we conducted cleavage assays using various type IIC Cas9 orthologs previously shown to function in human cells (Esvelt et al, 2013; Harrington et al, 2017; Hou et al, 2013; Kim et al, 2017). We found that in addition to NmeCas9, the AcrIIC1 from Neisseria meningitidis (AcrIIC1 Nme ) exhibits robust inhibition of the Cas9 proteins from Geobacillus stearothermophilus (GeoCas9) and Campylobacter jejuni (CjeCas9) (Figure 1B and S1B).…”

Section: Resultsmentioning

confidence: 99%

“…To determine which region of Cas9 interacts with AcrIIC1, we generated Cas9 truncations and tested their abilities to bind to AcrIIC1 using size exclusion chromatography (Figure 3A, 3B and S3A). Although many NmeCas9 truncations were insoluble, we took advantage of the thermostable GeoCas9 (Harrington et al, 2017) to generate soluble truncations. AcrIIC1 was able to associate with GeoCas9 without either the REC or PAM-interacting domains (Figure 3A), leaving the two nuclease domains as potential interacting partners.…”

Section: Resultsmentioning

confidence: 99%

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A Broad-Spectrum Inhibitor of CRISPR-Cas9

Harrington¹,

Doxzen²,

Ma³

et al. 2017

Cell

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SUMMARY CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off-switch for Cas9-based applications. Here we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs through direct binding to the conserved HNH catalytic domain of Cas9. A crystal structure of an AcrIIC1-Cas9 HNH domain complex shows how AcrIIC1 traps Cas9 in a DNA-bound but catalytically inactive state. By contrast, AcrIIC3 blocks activity of a single Cas9 ortholog and induces Cas9 dimerization while preventing binding to the target DNA. These two orthogonal mechanisms allow for separate control of Cas9 target binding and cleavage and suggest applications to allow DNA binding while preventing DNA cutting by Cas9.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

A Broad-Spectrum Inhibitor of CRISPR-Cas9

Harrington¹,

Doxzen²,

Ma³

et al. 2017

Cell

Self Cite

234

317

View full text Add to dashboard Cite

show abstract

“…Both the PAMs and the required guide-target base pairing for Type II-C Cas9s are longer than those of other subtypes, which make them inherently more specific (Mir et al, 2018). Genome editing in mammalian cells with three Type II-C Cas9s indeed showed a low off-target activity (Esvelt et al, 2013; Harrington et al, 2017; Hou et al, 2013; Kim et al, 2017; Lee, Cradick, & Bao, 2016). Additionally, many organisms that host Type II-C exist in extreme conditions such as Yellowstone national park acidic hot springs ( Acidothermus cellulolyticus ), pig respiratory tracts ( Pasteurella multocida ), and waste water land from Thailand ( Tisterilla mobilis ), and their Cas9s are stable and suited for genome editing under unusual conditions.…”

Section: Introductionmentioning

confidence: 99%

“…Additionally, many organisms that host Type II-C exist in extreme conditions such as Yellowstone national park acidic hot springs ( Acidothermus cellulolyticus ), pig respiratory tracts ( Pasteurella multocida ), and waste water land from Thailand ( Tisterilla mobilis ), and their Cas9s are stable and suited for genome editing under unusual conditions. For example, the Type II-C Cas9 from Geobacillus stearothermophilus (GeoCas9) has been shown to have a higher lifetime in human plasma making it a better tool as an RNP to inject in human bloodstream (Harrington et al, 2017). …”

Section: Introductionmentioning

confidence: 99%

Directed evolution studies of a thermophilic Type II-C Cas9

Hand

Das

2019

Methods in Enzymology

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Though making up nearly half of the known CRISPR–Cas9 family of enzymes, the Type II-C CRISPR–Cas9 has been underexplored for their molecular mechanisms and potential in safe gene editing applications. In comparison with the more popular Type II-A CRISPR–Cas9, the Type II-C enzymes are generally smaller in size and utilize longer base pairing in identification of their DNA substrates. These characteristics suggest easier portability and potentially less off-targets for Type II-C in gene editing applications. We describe identification and biochemical characterization of a thermophilic Type II-C CRISPR–Cas from Acidothermus cellulolyticus (AceCas9). We describe several library-based methods that enabled us to identify the PAM sequence and elements critical to protospacer mismatch surveillance of AceCas9

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“…There are also other natural CRISPR nucleases with completely different PAM sequences recognition shown to function efficiently in mammalian cells including Campylobacter jejuni ‐derived Cas9 (CjCas9; Kim et al, 2017), Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1; Zetsche et al, 2015), Staphylococcus aureus (SaCas9; Ran et al, 2015), Francisella novicida (FnCas9; H. Hirano et al, 2016), Streptococcus thermophilus Cas9 (StCas9; Müller et al, 2016), Geobacillus stearothermophilus (GeoCas9; Harrington et al, 2017), Neisseria meningitidis Cas9 (NmeCas9; Lee, Cradick, & Bao, 2016), Streptococcus canis (ScCas9; Chatterjee, Jakimo, & Jacobson, 2018). These CRISPR nucleases have extended the targeting range in CRISPR/Cas‐mediated genome editing.…”

Section: Introductionmentioning

confidence: 99%

Targeted gene disruption by CRISPR/xCas9 system in Drosophila melanogaster

Zhou

Huang

et al. 2020

Arch Insect Biochem Physiol

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Although the Cas9 protein from Streptococcus pyogenes (SpCas9) is the most widely used clustered regularly interspaced short palindromic repeats (CRISPR) variant in genome engineering experiments, it does have certain limitations. First, the stringent requirement for the protospacer adjacent motif (PAM) sequence limits the target DNA that can be manipulated using this method in insects. Second, its complementarity specifications are not very stringent, meaning that it can sometimes cause off‐target effects at the target site. A recent study reported that an evolved SpCas9 variant, xCas9(3.7), with preference for various 5′‐NG‐3′ PAM sequences not only has the broadest PAM compatibility but also has much greater DNA specificity and lower genome‐wide off‐target activity than SpCas9 in mammalian cells. Here we applied the CRISPR/xCas9 system to target the white gene in Drosophila melanogaster, testing the genome‐editing efficiency of xCas9 at different PAM sites. On the GGG PAM site, xCas9 showed less activity than SpCas9. For the non‐NGG PAM site TGA, xCas9 could produce DNA cleavage and indel‐mediated disruption on the target gene. However, for other non‐NGG PAM sites, xCas9 showed no activity. These findings show that the evolved Cas9 variant with broad PAM compatibility is functional in Drosophila to induce heritable gene alterations, increasing the targeting range for the applications of genome editing in insects.

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A thermostable Cas9 with increased lifetime in human plasma

Cited by 36 publications

References 43 publications

A Broad-Spectrum Inhibitor of CRISPR-Cas9

A Broad-Spectrum Inhibitor of CRISPR-Cas9

Directed evolution studies of a thermophilic Type II-C Cas9

Targeted gene disruption by CRISPR/xCas9 system in Drosophila melanogaster

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