Catalytically Enhanced Cas9 Through Directed Protein Evolution

Hand, Travis H.; Roth, Mitchell O.; Smith, Chardasia L.; Shiel, Emily; Klein, Kyle N.; Gilbert, David M.; Li, Hong

doi:10.1089/crispr.2020.0092

Cited by 14 publications

(15 citation statements)

References 42 publications

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“…CRISPR/Cas9 gene editing system has been widely used in basic cancer research. By specifically correcting mutations, some progress has been made in inhibiting the growth and progression of colon cancer ( 75 , 76 ).…”

Section: Crispr/cas9 Gene Editingmentioning

confidence: 99%

Application of CRISPR-Cas9 gene editing technology in basic research, diagnosis and treatment of colon cancer

Meng

Nan

et al. 2023

Front. Endocrinol.

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Colon cancer is the fourth leading cause of cancer death worldwide, and its progression is accompanied by a complex array of genetic variations. CRISPR/Cas9 can identify new drug-resistant or sensitive mutations in colon cancer, and can use gene editing technology to develop new therapeutic targets and provide personalized treatments, thereby significantly improving the treatment of colon cancer patients. CRISPR/Cas9 systems are driving advances in biotechnology. RNA-directed Cas enzymes have accelerated the pace of basic research and led to clinical breakthroughs. This article reviews the rapid development of CRISPR/Cas in colon cancer, from gene editing to transcription regulation, gene knockout, genome-wide CRISPR tools, therapeutic targets, stem cell genomics, immunotherapy, metabolism-related genes and inflammatory bowel disease. In addition, the limitations and future development of CRISPR/Cas9 in colon cancer studies are reviewed. In conclusion, this article reviews the application of CRISPR-Cas9 gene editing technology in basic research, diagnosis and treatment of colon cancer.

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Section: Crispr/cas9 Gene Editingmentioning

confidence: 99%

Application of CRISPR-Cas9 gene editing technology in basic research, diagnosis and treatment of colon cancer

Meng

Nan

et al. 2023

Front. Endocrinol.

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“…The copyright holder for this preprint this version posted September 11, 2022. ; https://doi.org/10.1101/2022.09. 10.507422 doi: bioRxiv preprint 4 HNH conformational placement 5,[7][8][9] . Correctly placed HNH, but not necessarily its catalytic activity, activates the RuvC domain for the cleavage of the nontarget DNA strand 10 .…”

Section: Introductionmentioning

confidence: 99%

“…AceCas9 is moderately thermophilic and specific to DNA protospacers associated with a 5’-NNNCC-3’ PAM. Though the wild-type AceCas9 has slower rate of cleavage than that of SpyCas9, protein directed engineering has resulted in a catalytic enhanced AceCas9 that has a comparable cleavage rate to that of SpyCas9 9 . Importantly, AceCas9 is uniquely sensitive to DNA methylation and does not cleave DNA with a 5’-NNN m CC-3’ PAM 16 .…”

Section: Introductionmentioning

confidence: 99%

“…Concomitant with the engagement of the REC lobe with the guide-DNA heteroduplex, the HNH domain undergoes a large conformational change to be docked onto the target strand 5,6 . Mutations in REC, the RuvC-HNH linker or the guide-DNA heteroduplex that are known to change catalytic efficiencies also impact the rate of 4 HNH conformational placement 5,[7][8][9] . Correctly placed HNH, but not necessarily its catalytic activity, activates the RuvC domain for the cleavage of the nontarget DNA strand 10 .…”

Section: Introductionmentioning

confidence: 99%

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Conformational Changes Regulate Metal Coordination in the Catalytic Sites of Cas9

Das

Rai

Roth

et al. 2022

Preprint

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The control of CRISPR-Cas9 activities plays an essential role in its safe adoption for clinical and research applications. Previous studies have identified conformational dynamics of Cas9 as a key regulatory element for its enzymatic activity. The molecular basis for how conformations influence the catalytic processes at both nuclease domains, however, remains elusive. Here we present well-resolved cryo-EM structures of the active Acidothermus cellulolyticus Cas9 (AceCas9) complexes representing pre-cleavage (2.9 &Aring), two reaction intermediate (2.2 &Aring and 2.4 &Aring) and two post-cleavage (2.6 &Aring and 2.7 &Aring) states that reveal active site rearrangement during DNA binding and phosphodiester bond breakage by Cas9. Strikingly, large domain rearrangements in Cas9 triggered by the cognate DNA result in subtle changes in active sites that facilitate coordination of metal ions required for catalysis. The two reaction intermediate structures further reveal small oscillations in domain conformations, which alternates the reactive states of the two catalytic centers, thereby coupling cleavage of the two DNA strands. Consistent with the roles of conformations in organizing the active sites, adjustments of metal coordination ligands lead to altered metal specificity.

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“…These assays rely on toxins encoded on target plasmids that are cured by endonuclease activity to rescue growth, linking cell survival to nuclease activity. However, common toxins such as ccdB − and barnase are highly lethal. Low levels of leaky expression can cause cell death reducing sensitivity to enzymes with low activity levels but otherwise desirable properties such as substrate preference .…”

mentioning

confidence: 99%

Repurposing the Homing Endonuclease I-SceI for Positive Selection and Development of Gene-Editing Technologies

et al. 2022

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Prokaryote genomes encode diverse programmable DNA endonucleases with significant potential for biotechnology and gene editing. However, these endonucleases differ significantly in their properties, which must be screened and measured. While positive selection screens based on ccdB and barnase have been developed to evaluate such proteins, their high levels of toxicity make them challenging to use. Here, we develop and validate a more robust positive selection screen based on the homing endonuclease I-SceI. Candidate endonucleases target and cure the I-SceI expression plasmid preventing induction of I-SceI-mediated double strand DNA breaks that lead to cell death in E. coli. We validated this screen to measure the relative activity of SpCas9, xCas9, and eSpCas9 and demonstrated an ability to enrich for more active endonuclease variants from a mixed population. This system may be applied in high throughput to rapidly characterize novel programmable endonucleases and be adapted for directed evolution of endonuclease function.

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Catalytically Enhanced Cas9 Through Directed Protein Evolution

Cited by 14 publications

References 42 publications

Application of CRISPR-Cas9 gene editing technology in basic research, diagnosis and treatment of colon cancer

Application of CRISPR-Cas9 gene editing technology in basic research, diagnosis and treatment of colon cancer

Conformational Changes Regulate Metal Coordination in the Catalytic Sites of Cas9

Repurposing the Homing Endonuclease I-SceI for Positive Selection and Development of Gene-Editing Technologies

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