A catalogue of 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:10.1038/s41467-020-19344-1

Cited by 133 publications

(167 citation statements)

References 74 publications

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“…In this study, we con rmed that the FnCas9 nickase formed a nick 6-8 bp from the PAM on the non-target strand side, and extended the editable region by prime editor. This effect could be also applicable to other endonucleases that induce the nick formation in different fashion [21], and in particular, the farther the nick is generated upstream from the PAM, the wider the range of target expansion will be. In addition to this, the FnCas9(H969A)-RT based editable region could be further extended by engineering the PAM recognition domain within the FnCas9 nickase as previous study [20].…”

Section: Resultsmentioning

confidence: 99%

Expansion of the Prime editing Modality with Cas9 from Francisella novicida

Lee

et al. 2021

Preprint

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Prime editing can induce a desired base substitution, insertion, or deletion in a target gene using reverse transcriptase (RT) after nick formation by CRISPR nickase. In this study, we developed a technology that can be used to insert or replace external bases in the target DNA sequence by linking reverse transcriptase to the Francisella novicida Cas9, which is a CRISPR-Cas9 ortholog. Using FnCas9(H969A) nickase, the targeting limitation of existing Streptococcus pyogenes Cas9 nickase [SpCas9(H840A)]-based prime editing was dramatically extended, and accurate prime editing was induced specifically for the target genes in human cell line.

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Section: Resultsmentioning

confidence: 99%

Expansion of the Prime editing Modality with Cas9 from Francisella novicida

Lee

et al. 2021

Preprint

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“…Due to being the first ortholog discovered, there has been a much greater exploration of Cas9 ortholog diversity and re-engineering of successful orthologs into higher activity variants than for effectors from other types [ 80 ] ( Table 1 ) [ 27 , 46 – 47 , 81 , 101 – 103 ] . The driving motivation for using different Cas9 orthologs for editing lies in their different PAM requirements, protein size to fit into a delivery vector and editing efficacy at different target sites in different organisms.…”

Section: Class 2 Effectors: Monomeric Endonucleases Which Form the Cmentioning

confidence: 99%

Harnessing CRISPR-Cas system diversity for gene editing technologies

McKay¹,

Burgio²

2021

J Biomed Res

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The discovery and utilization of RNA-guided surveillance complexes, such as CRISPR-Cas9, for sequence-specific DNA or RNA cleavage, has revolutionised the process of gene modification or knockdown. To optimise the use of this technology, an exploratory race has ensued to discover or develop new RNA-guided endonucleases with the most flexible sequence targeting requirements, coupled with high cleavage efficacy and specificity. Here we review the constraints of existing gene editing and assess the merits of exploiting the diversity of CRISPR-Cas effectors as a methodology for surmounting these limitations.

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“…The number of sgRNAs that can be designed in the vicinity of certain genomic regions is low. To broaden the accessibility of DNA sequences for the CRISPR system, Cas9 variants and orthologs, as well as other Cas proteins, can be used to recognize diverse PAM sequences [28,[36][37][38]. For instance, xCas9 can recognize NG, GAA and GAT PAMs [39], while SpCas9-NG can recognize relaxed NG PAMs [40].…”

Section: Limitations and Future Prospectsmentioning

confidence: 99%

Full-Spectrum Targeted Mutagenesis in Plant and Animal Cells

Iaffaldano

Reiser

2021

IJMS

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Directed evolution is a powerful approach for protein engineering and functional studies. However, directed evolution outputs from bacterial and yeast systems do not always translate to higher organisms. In situ directed evolution in plant and animal cells has previously been limited by an inability to introduce targeted DNA sequence diversity. New hypermutation tools have emerged that can generate targeted mutations in plant and animal cells, by recruiting mutagenic proteins to defined DNA loci. Progress in this field, such as the development of CRISPR-derived hypermutators, now allows for all DNA nucleotides within user-defined regions to be altered through the recruitment of error-prone DNA polymerases or highly active DNA deaminases. The further engineering of these mutagenesis systems will potentially allow for all transition and transversion substitutions to be generated within user-defined genomic windows. Such targeted full-spectrum mutagenesis tools would provide a powerful platform for evolving antibodies, enzymes, structural proteins and RNAs with specific desired properties in relevant cellular contexts. These tools are expected to benefit many aspects of biological research and, ultimately, clinical applications.

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A catalogue of biochemically diverse CRISPR-Cas9 orthologs

Cited by 133 publications

References 74 publications

Expansion of the Prime editing Modality with Cas9 from Francisella novicida

Expansion of the Prime editing Modality with Cas9 from Francisella novicida

Harnessing CRISPR-Cas system diversity for gene editing technologies

Full-Spectrum Targeted Mutagenesis in Plant and Animal Cells

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