High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs

Huang, Tony P.; Heins, Zachary J.; Miller, Shannon M.; Wong, Brandon G.; Balivada, Pallavi A.; Wang, Tina; Khalil, Ahmad S.; Liu, David R.

doi:10.1038/s41587-022-01410-2

Cited by 61 publications

(30 citation statements)

References 60 publications

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“…Directed evolution combined with in vitro high-throughput screening is a powerful method for the generation of mutant proteins with improved functional properties or the achievement of tolerance to strict reaction conditions ( Huang et al, 2023 ). High genetic diversity in a target gene can be generated by epPCR through random base substitutions during DNA replication; this approach is widely used in experimental analyses.…”

Section: Resultsmentioning

confidence: 99%

Thermostability enhancement of Escherichia coli phytase by error-prone polymerase chain reaction (epPCR) and site-directed mutagenesis

Xing

Wang

Yan

et al. 2023

Front. Bioeng. Biotechnol.

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Phytase efficiently hydrolyzes phytate to phosphate; thus, it is widely used to increase phosphorus availability in animal feeds and reduce phosphorus pollution through excretion. Phytase is easily inactivated during feed pelleting at high temperature, and sufficient thermostability of phytase is essential for industrial applications. In this study, directed evolution was performed to enhance phytase thermostability. Variants were initially expressed in Escherichia coli BL21 for screening, then in Pichia pastoris for characterization. Over 19,000 clones were generated from an error-prone Polymerase Chain Reaction (epPCR) library; 5 mutants (G10, D7, E3, F8, and F9) were obtained with approximately 9.6%, 10.6%, 11.5%, 11.6%, and 12.2% higher residual activities than the parent after treatment at 99°C for 60 min. Three of these mutants, D7, E3, and F8, exhibited 79.8%, 73.2%, and 92.6% increases in catalytic efficiency (kcat/Km), respectively. In addition, the specific activities of D7, E3, and F8 were 2.33-, 1.98-, and 2.02-fold higher than parental phytase; they were also higher than the activities of all known thermostable phytases. Sequence analysis revealed that all mutants were substituted at residue 75 and was confirmed that the substitution of cysteine at position 75 was the main contribution to the improvement of thermostability of mutants by saturation mutagenesis, indicating that this amino acid is crucial for the stability and catalytic efficiency of phytase. Docking structure analysis revealed that substitution of the C75 residue allowed the mutants to form additional hydrogen bonds in the active pocket, thereby facilitating binding to the substrate. In addition, we confirmed that the intrinsic C77-C108 disulfide bond in E. coli phytase is detrimental to its stability.

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Section: Resultsmentioning

confidence: 99%

Thermostability enhancement of Escherichia coli phytase by error-prone polymerase chain reaction (epPCR) and site-directed mutagenesis

Xing

Wang

Yan

et al. 2023

Front. Bioeng. Biotechnol.

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“…Yet, many bioassays contain single inputs and can hardly be adapted to other purposes. Here, we show that it is possible to generate logic gates using a split CRISPR/Cas9, which is an actuator with many downstream options [61][62][63][64] . Moreover, the activation of Cas9 also creates a memory so using this system we could detect biological processes after they occurred e.g.…”

Section: Detection Of Cell-cell Fusion Eventsmentioning

confidence: 98%

Synthetic Circuits Based on Split Cas9 to Detect Cellular Events

Przybyszewska-Podstawka

Czapiński

Kałafut

et al. 2023

Preprint

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Synthetic biology involves the generation of logic circuits to create or control biological functions and behaviours by engineering interconnected genetic elements such as promoters, repressors, and transcriptional activators. CRISPR discovery, and its adaptations to mammalian cells, has made it the tool of choice in molecular biology and revolutionized genome engineering in biomedical sciences. Here, we describe an adaptation of a split Cas9 to generate synthetic logic gates to sense biological events. As proof-of-concept, the complementing halves of split Cas9 were placed under different promoters, one unique to cancer cells of epithelial origin (phCEA) and one universal promoter (pCMV). We used self-assembling inteins to reunite the halves when co-expressed. Only cancer cells with epithelial origin expressed both halves and activated a reporter becoming green fluorescent. We then investigated whether we could apply this system to the detection of biological processes such as epithelial to mesenchymal transition (EMT). We designed another logic gate where one halve is expressed only by cancer cells of epithelial origin, while the other is activated during EMT - under the control of TWIST1. Indeed, cells undergoing EMT were detected by the activation of the reporter. Finally, the split-Cas9 logic gate was applied as a sensor to detect cell-cell fusion events in multiple cell lines. Each cell type expressed only one halve of split Cas9, and only induction of fusion resulted in the appearance of multinucleated syncytia and the expression of the reporter system. The simplicity and flexibility of the split Cas9 system reported here can be integrated to many other cellular processes, not only as a sensor but as an actuator.

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“…[98][99][100] Due to the limitations of the PAM, the CRISPR/Cas9 gene-editing system often fails to target the proper sites. Therefore, the modification of Cas9 focuses on two goals: enhancing the security of Cas9 [101][102][103][104][105][106][107][108][109][110] and freeing it from the limitations of PAM [111][112][113][114][115][116][117] (Tables 1, 2). Method for CRISPR delivery Plasmid DNA (pDNA) is an ideal vector for loading the CRISPR system because it is not easily degradable, can be amplified in large quantities, and can be easily modified.…”

Section: Composition Of Crispr/cas9mentioning

confidence: 99%

CRISPR/Cas9 therapeutics: progress and prospects

Yang

et al. 2023

Sig Transduct Target Ther

223

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Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene-editing technology is the ideal tool of the future for treating diseases by permanently correcting deleterious base mutations or disrupting disease-causing genes with great precision and efficiency. A variety of efficient Cas9 variants and derivatives have been developed to cope with the complex genomic changes that occur during diseases. However, strategies to effectively deliver the CRISPR system to diseased cells in vivo are currently lacking, and nonviral vectors with target recognition functions may be the focus of future research. Pathological and physiological changes resulting from disease onset are expected to serve as identifying factors for targeted delivery or targets for gene editing. Diseases are both varied and complex, and the choice of appropriate gene-editing methods and delivery vectors for different diseases is important. Meanwhile, there are still many potential challenges identified when targeting delivery of CRISPR/Cas9 technology for disease treatment. This paper reviews the current developments in three aspects, namely, gene-editing type, delivery vector, and disease characteristics. Additionally, this paper summarizes successful examples of clinical trials and finally describes possible problems associated with current CRISPR applications.

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High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs

Cited by 61 publications

References 60 publications

Thermostability enhancement of Escherichia coli phytase by error-prone polymerase chain reaction (epPCR) and site-directed mutagenesis

Thermostability enhancement of Escherichia coli phytase by error-prone polymerase chain reaction (epPCR) and site-directed mutagenesis

Synthetic Circuits Based on Split Cas9 to Detect Cellular Events

CRISPR/Cas9 therapeutics: progress and prospects

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