Computationally designed hyperactive Cas9 enzymes

Vos, Pascal D.; Rossetti, Giulia; Mantegna, Jessica L.; Siira, Stefan J.; Gandadireja, Andrianto P.; Bruce, Mitchell; Raven, Samuel A.; Khersonsky, Olga; Fleishman, Sarel J.; Filipovska, Aleksandra; Rackham, Oliver

doi:10.1038/s41467-022-30598-9

Cited by 16 publications

(5 citation statements)

References 39 publications

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“…Through our structural analysis, we identified a mutation that accelerates gRNA:TS duplex formation and increases DNA cleavage efficiency (Fig 3). Combining this mutation with other hyperactive Cas9 mutations holds the potential to compensate for SpRY's reduced activity (38). The creation of chimeric CRISPR-Cas9 variants, where mutants with diverse purposes are combined, could forge the development of highly specific and adaptable gene editing tools that transcend biological constraints.…”

Section: Discussionmentioning

confidence: 99%

Unraveling the mechanisms of PAMless DNA interrogation by SpRY Cas9

Hibshman

Bravo

Zhang

et al. 2023

Preprint

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CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately adjacent to the DNA target limits the number of editable genes. To overcome the PAM requirement, a recently developed Cas9 variant, called SpRY-Cas9 was engineered to be PAMless. However, the molecular mechanisms of how SpRY can recognize all potential PAM sequences and still accurately identify DNA targets have not been investigated. Here, we combined enzyme kinetics, cryo-EM, and single-molecule imaging to determine how SpRY interrogates DNA and recognizes target sites for cleavage. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region of SpRY, which facilitates tight binding to off-target DNA sequences. Once SpRY correctly identifies a target site, nuclease activation occurs ~1,000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Insights gained from our intermediate structures prompted rationally designed mutants with improved DNA cleavage efficiency. Our findings shed light on the molecular mechanisms of PAMless genome editing with SpRY and provide a framework for the design of future genome editing tools with improved versatility, precision, and efficiency.

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Section: Discussionmentioning

confidence: 99%

Unraveling the mechanisms of PAMless DNA interrogation by SpRY Cas9

Hibshman

Bravo

Zhang

et al. 2023

Preprint

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“…MD was also used to gain insight into DNA/RNA binding, [341] enzyme activation via domain rearrangements, [342] and to unravel the effect of mutations in high‐fidelity Cas9 variants [343] . The FuncLib tool allowed identification of highly active Cas9 variants, [344] and various ancestral Cas9 variants were created that accept different PAMs and guide RNA backbones [345] . Parameterizing the non‐proteogenic parts in Cas9 for MD required some additional care: for MDs, DNA and RNA were generally simulated using either the Parmbsc0 or Parmbsc1 force field refinements, while the magnesium ions were usually modelled with refined non‐bonded force field parameters [339–340,342] …”

Section: Examplesmentioning

confidence: 99%

Curse and Blessing of Non‐Proteinogenic Parts in Computational Enzyme Engineering

Korbeld

Fürst

2023

ChemBioChem

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Enzyme engineering aims to improve or install a new function in biocatalysts for applications ranging from chemical synthesis to biomedicine. For decades, computational techniques have been developed to predict the effect of protein changes and design new enzymes. However, these techniques may have been optimized to deal with proteins composed of the standard amino acid alphabet, while the function of many enzymes relies on non‐proteogenic parts like cofactors, nucleic acids, and post‐translational modifications. Enzyme systems containing such molecules might be handled or modeled improperly by computational tools, and thus be unsuitable, or require additional tweaking, parameterization, or preparation. In this review, we give an overview of common and recent tools and workflows available to computational enzyme engineers. We highlight the various pitfalls that come with including non‐proteogenic compounds in computations and outline potential ways to address common issues. Finally, we showcase successful examples from the literature that computationally engineered such enzymes.

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“…This technology uses plasmids to introduce the Cas9 enzyme and guide RNA molecules into a host cell. [ 61 ] Once inside the cell, the Cas9 enzyme can cut the host cell's DNA at specific locations, allowing researchers to add, delete, or modify genes. In synthetic biology, Plasmid is used to create new biological systems and organisms.…”

Section: Nucleic Acids Nanotechnologymentioning

confidence: 99%

Harnessing Nucleic Acids Nanotechnology for Bone/Cartilage Regeneration

Han

Cao²,

et al. 2023

Small

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The effective regeneration of weight‐bearing bone defects and critical‐sized cartilage defects remains a significant clinical challenge. Traditional treatments such as autologous and allograft bone grafting have not been successful in achieving the desired outcomes, necessitating the need for innovative therapeutic approaches. Nucleic acids have attracted significant attention due to their ability to be designed to form discrete structures and programmed to perform specific functions at the nanoscale. The advantages of nucleic acid nanotechnology offer numerous opportunities for in‐cell and in vivo applications, and hold great promise for advancing the field of biomaterials. In this review, the current abilities of nucleic acid nanotechnology to be applied in bone and cartilage regeneration are summarized and insights into the challenges and future directions for the development of this technology are provided.

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Computationally designed hyperactive Cas9 enzymes

Cited by 16 publications

References 39 publications

Unraveling the mechanisms of PAMless DNA interrogation by SpRY Cas9

Unraveling the mechanisms of PAMless DNA interrogation by SpRY Cas9

Curse and Blessing of Non‐Proteinogenic Parts in Computational Enzyme Engineering

Harnessing Nucleic Acids Nanotechnology for Bone/Cartilage Regeneration

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