Improving the on-target activity of high-fidelity Cas9 editors by combining rational design and random mutagenesis

Spasskaya, D. S.; Давлетшин, А. И.; Bachurin, S. S.; Tutyaeva, Vera V.; Гарбуз, Д. Г.; Карпов, Д. С.

doi:10.1007/s00253-023-12469-5

Cited by 7 publications

(33 citation statements)

References 66 publications

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“…The first variant to show increased activity in the context of yeast chromatin was iCas9 carrying D147Y and P411T mutations in the REC-I domain (Bao et al 2015 ). Our work shows that the addition of iCas9 mutations generally increases the activity of high-fidelity variants (Spasskaya et al 2023 ). However, off-target activity is also increased.…”

Section: Next-generation High-fidelity Cas9 Variants With Increased A...mentioning

confidence: 55%

“…Another way for improving first-generation Cas9 editors was implemented in our recent work by adding a novel L1206P mutation in PID (Spasskaya et al 2023 ). The mutation position has not been previously identified in any work, including the comprehensive Cas9 mutagenesis screening (Spencer and Zhang 2017 ).…”

Section: Next-generation High-fidelity Cas9 Variants With Increased A...mentioning

confidence: 99%

“…A growing body of evidence suggests that the nucleosome presents a barrier to genome editing (Verkuijl and Rots 2019 ; Dubois 2022 ). This barrier appears to be higher for high-fidelity Cas9 variants (X. Chen et al 2017a , b ; Spasskaya et al 2023 ). Therefore, several ways to overcome this barrier are being explored, including recruiting a transcriptional machinery (Liu et al 2019 ; Daer et al 2020 ), adding DNA- or histone-binding proteins and peptides (Ding et al 2019 ), or using inhibitors of chromatin-modifying complexes (B. Liu et al 2020a , b ; J. P. Zhang et al 2021a , b , c ).…”

Section: Next-generation High-fidelity Cas9 Variants With Increased A...mentioning

confidence: 99%

“…Another pair of R221K and N394K mutations in the REC-II and REC-I domains, respectively, increased the activity of the chimeric iSpyMAC (a Cas9-based editor for the AA dinucleotide PAM) (Chatterjee et al 2020a , b ). The L1206P or A1345L mutations also enhance the activity of wild-type Cas9 and its high-fidelity derivatives on nucleosomes (Spasskaya et al 2023 ; Davletshin et al 2024 ). Clearly, Cas9 mutations in different domains suggest that the nucleosome barrier can be overcome by different mechanisms.…”

Section: Next-generation High-fidelity Cas9 Variants With Increased A...mentioning

confidence: 99%

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Engineering Cas9: next generation of genomic editors

Kovalev,

Davletshin,

Karpov

2024

Appl Microbiol Biotechnol

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The Cas9 endonuclease of the CRISPR/Cas type IIA system from Streptococcus pyogenes is the heart of genome editing technology that can be used to treat human genetic and viral diseases. Despite its large size and other drawbacks, S. pyogenes Cas9 remains the most widely used genome editor. A vast amount of research is aimed at improving Cas9 as a promising genetic therapy. Strategies include directed evolution of the Cas9 protein, rational design, and domain swapping. The first generation of Cas9 editors comes directly from the wild-type protein. The next generation is obtained by combining mutations from the first-generation variants, adding new mutations to them, or refining mutations. This review summarizes and discusses recent advances and ways in the creation of next-generation genomic editors derived from S. pyogenes Cas9. Key points • The next-generation Cas9-based editors are more active than in the first one. • PAM-relaxed variants of Cas9 are improved by increased specificity and activity. • Less mutagenic and immunogenic variants of Cas9 are created.

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Section: Next-generation High-fidelity Cas9 Variants With Increased A...mentioning

confidence: 55%

Section: Next-generation High-fidelity Cas9 Variants With Increased A...mentioning

confidence: 99%

Section: Next-generation High-fidelity Cas9 Variants With Increased A...mentioning

confidence: 99%

Section: Next-generation High-fidelity Cas9 Variants With Increased A...mentioning

confidence: 99%

See 2 more Smart Citations

Engineering Cas9: next generation of genomic editors

Kovalev,

Davletshin,

Karpov

2024

Appl Microbiol Biotechnol

Self Cite

View full text Add to dashboard Cite

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“…In attempts to generate both compact and broad effector Cas proteins, scientists have deepened the search into Cas orthologs in a bid to identify Cas effectors that are small and have PAM sites that allow for more flexibility ( Esvelt et al, 2013 ; Shmakov et al, 2015 ; Burstein et al, 2017 ; Shmakov et al, 2017 ; Harrington et al, 2018 ; Gasiunas et al, 2020 ; Hu et al, 2020 ; Wang et al, 2020 ). To this end, variants of the Cas proteins have been engineered through base-editing the amino acids around the PAM recognition domain to enhance the efficiency of the Cas protein, such as HypaCas9 and eSpCas9 ( Slaymaker et al, 2016 ; Tycko et al, 2016 ; Schmid-Burgk et al, 2020 ), or to alter the PAM recognition site of the Cas effector, such as SpCas9-NG or SaCas9-KKH which recognize NG and NNNRRT, respectively ( Kleinstiver et al, 2015a ; Kleinstiver et al, 2015b ; Anders et al, 2016 ; Hirano et al, 2016b ; Gao et al, 2017 ; Hu et al, 2018 ; Yuen et al, 2022 ; Spasskaya et al, 2023 ). Therefore, the Cas system has significantly expanded as a toolbox for targeting DNA or RNA and these orthologs and variants of Cas effectors have yet to be fully explored for their use in targeting the HIV-1 provirus.…”

Section: Introductionmentioning

confidence: 99%

Computational analysis of cas proteins unlocks new potential in HIV-1 targeted gene therapy

Dampier,

Berman,

Nonnemacher

et al. 2024

Front. Genome Ed.

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Introduction: The human immunodeficiency virus type 1 (HIV-1) pandemic has been slowed with the advent of anti-retroviral therapy (ART). However, ART is not a cure and as such has pushed the disease into a chronic infection. One potential cure strategy that has shown promise is the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas gene editing system. It has recently been shown to successfully edit and/or excise the integrated provirus from infected cells and inhibit HIV-1 in vitro, ex vivo, and in vivo. These studies have primarily been conducted with SpCas9 or SaCas9. However, additional Cas proteins are discovered regularly and modifications to these known proteins are being engineered. The alternative Cas molecules have different requirements for protospacer adjacent motifs (PAMs) which impact the possible targetable regions of HIV-1. Other modifications to the Cas protein or gRNA handle impact the tolerance for mismatches between gRNA and the target. While reducing off-target risk, this impacts the ability to fully account for HIV-1 genetic variability.Methods: This manuscript strives to examine these parameter choices using a computational approach for surveying the suitability of a Cas editor for HIV-1 gene editing. The Nominate, Diversify, Narrow, Filter (NDNF) pipeline measures the safety, broadness, and effectiveness of a pool of potential gRNAs for any PAM. This technique was used to evaluate 46 different potential Cas editors for their HIV therapeutic potential.Results: Our examination revealed that broader PAMs that improve the targeting potential of editors like SaCas9 and LbCas12a have larger pools of useful gRNAs, while broader PAMs reduced the pool of useful SpCas9 gRNAs yet increased the breadth of targetable locations. Investigation of the mismatch tolerance of Cas editors indicates a 2-missmatch tolerance is an ideal balance between on-target sensitivity and off-target specificity. Of all of the Cas editors examined, SpCas-NG and SPRY-Cas9 had the highest number of overall safe, broad, and effective gRNAs against HIV.Discussion: Currently, larger proteins and wider PAMs lead to better targeting capacity. This implies that research should either be targeted towards delivering longer payloads or towards increasing the breadth of currently available small Cas editors. With the discovery and adoption of additional Cas editors, it is important for researchers in the HIV-1 gene editing field to explore the wider world of Cas editors.

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