Biochemically diverse CRISPR-Cas9 orthologs

Gasiūnas, Giedrius; Young, Joshua K.; Karvelis, Tautvydas; Kazlauskas, Darius; Urbaitis, Tomas; Jasnauskaite, Monika; Grusyte, Mantvyda M.; Paulraj, Sushmitha; Wang, Po-Hao; Hou, Zhenglin; Dooley, Shane K.; Cigan, Mark; Alarcon, Clara M.; Chilcoat, N. Doane; Bigelyte, Greta; Curcuru, Jennifer L.; Mabuchi, Megumu; Sun, Zhiyi; Fuchs, Ryan T.; Schildkraut, Ezra; Weigele, Peter; Jack, William E.; Robb, G. Brett; Venclovas, C̆eslovas; Šikšnys, Virginijus

doi:10.1101/2020.04.29.066654

Cited by 8 publications

(4 citation statements)

References 94 publications

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“…Towards library design, in order to narrow down the vast mutational space associated with combinatorial libraries, we utilize an approach guided by evolution and natural variation (36,37). As deimmunizing protein engineering seeks to alter the amino acid sequence of a protein without disrupting functionality, it is extremely useful to narrow down mutations to those less likely to result in non-functional variants.…”

Section: Resultsmentioning

confidence: 99%

Extensive in vitro and in vivo protein translation via in situ circularized RNAs

Kumar

Palmer

Miyasaki

et al. 2022

Preprint

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RNAs are a powerful therapeutic class. However their inherent transience impacts their activity both as an interacting moiety as well as a template. Circularization of RNA has been demonstrated as a means to improve persistence, however simple and scalable approaches to achieve this are lacking. Utilizing autocatalytic RNA circularization, here we engineer in situ circularized RNAs (icRNAs). This approach enables icRNA delivery as simple linear RNA that is circularized upon delivery into the cell, thus making them compatible with routine synthesis, purification, and delivery formulations. We confirmed extensive protein translation from icRNAs both in vitro and in vivo and explored their utility in three contexts: first, we delivered the SARS-CoV-2 Omicron spike protein in vivo as icRNAs and showed corresponding induction of humoral immune responses; second, we demonstrated robust genome targeting via zinc finger nucleases delivered as icRNAs; and third, to enable compatibility between persistence of expression and immunogenicity, we developed a novel long range multiplexed (LORAX) protein engineering methodology to screen progressively deimmunized Cas9 proteins, and demonstrated efficient genome and epigenome targeting via their delivery as icRNAs. We anticipate this highly simple and scalable icRNA methodology could have broad utility in basic science and therapeutic applications.

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Section: Resultsmentioning

confidence: 99%

Extensive in vitro and in vivo protein translation via in situ circularized RNAs

Kumar

Palmer

Miyasaki

et al. 2022

Preprint

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“…Many Cas9 orthologs (e.g., ScCas9 [177], CjCas9 [333], NmeCas9 [334], FnCas9 [335], Smac-Cas9 [336], SauriCas9 [337], St1Cas9 [338], St3Cas9 [339], FrCas9 [76], and so on [340][341][342][343][344][345][346][347][348][349][350][351][352][353][354][355][356][357][358]) have been reported to be intrinsically high-fidelity [76,77], and some of them were engineered with a broad PAM range, high fidelity, and/or both [359] (Supplementary Table S4).…”

Section: Cas9 Orthologsmentioning

confidence: 99%

CRISPR/Cas9 Landscape: Current State and Future Perspectives

Tyumentseva,

Tyumentsev,

Akimkin

2023

IJMS

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CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is a unique genome editing tool that can be easily used in a wide range of applications, including functional genomics, transcriptomics, epigenetics, biotechnology, plant engineering, livestock breeding, gene therapy, diagnostics, and so on. This review is focused on the current CRISPR/Cas9 landscape, e.g., on Cas9 variants with improved properties, on Cas9-derived and fusion proteins, on Cas9 delivery methods, on pre-existing immunity against CRISPR/Cas9 proteins, anti-CRISPR proteins, and their possible roles in CRISPR/Cas9 function improvement. Moreover, this review presents a detailed outline of CRISPR/Cas9-based diagnostics and therapeutic approaches. Finally, the review addresses the future expansion of genome editors’ toolbox with Cas9 orthologs and other CRISPR/Cas proteins.

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“…Additionally, we visualized CasPEDIA's enzymatic classification efforts against the current genetic classification of Class 2 CRISPR systems (3,8). Phylogenetic trees were constructed for each Class 2 Type from comprehensive datasets for Cas9, Cas12, and Cas13 proteins (3,(17)(18)(19)(20). Trees were constructed with IQ-TREE from MUSCLE aligned sequences, and visualized in iTOL (21)(22)(23).…”

Section: Caspedia Data Curationmentioning

confidence: 99%

CasPEDIA Database: A Functional Classification System for Class 2 CRISPR-Cas Enzymes

Adler,

Trinidad,

Bellieny-Rabelo

et al. 2023

Preprint

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CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

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Biochemically diverse CRISPR-Cas9 orthologs

Cited by 8 publications

References 94 publications

Extensive in vitro and in vivo protein translation via in situ circularized RNAs

Extensive in vitro and in vivo protein translation via in situ circularized RNAs

CRISPR/Cas9 Landscape: Current State and Future Perspectives

CasPEDIA Database: A Functional Classification System for Class 2 CRISPR-Cas Enzymes

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