Beyond Native Cas9: Manipulating Genomic Information and Function

Mitsunobu, Hitoshi; Teramoto, Jun; Nishida, Keiji; Kondo, Akihiko

doi:10.1016/j.tibtech.2017.06.004

Cited by 64 publications

(39 citation statements)

References 88 publications

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“…Given their small size, DNA barcodes generally only minimally disrupt a locus but this needs to be verified for the locus of interest, as is the case for strategies involving targeting affinity handles by Cas9 or other approaches. Fortunately, genomic barcoding has come within reach for many research questions due to the availability emerging genome engineering strategies, in yeast and other organisms [63][64][65][66] . For example, in budding yeast, CRIPSR-Cas9 tools provide the means to integrate barcodes from simple oligonucleotide-derived repair templates with very high efficiency and precision without the need for a selectable marker.…”

Section: Discussionmentioning

confidence: 99%

Decoding the chromatin proteome of a single genomic locus by DNA sequencing

Korthout

Poramba‐Liyanage

Verzijlbergen

et al. 2017

Preprint

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Transcription, replication and repair involve interactions of specific genomic loci with many different proteins. How these interactions are orchestrated at any given location and under changing cellular conditions is largely unknown because systematically measuring protein-DNA interactions at a specific locus in the genome is challenging. To address this problem, we developed Epi-Decoder, a Tag-ChIPBarcode-Seq technology in budding yeast to identify and quantify in an unbiased and systematic manner the proteome of an individual genomic locus. Epi-Decoder is orthogonal to proteomics approaches because it does not rely on mass spectrometry but instead takes advantage of DNA sequencing. Analysis of the proteome of a transcribed locus proximal to an origin of replication revealed more than 400 proteins. Moreover, replication stress induced changes in local chromatin-proteome composition prior to local origin firing, affecting replication proteins as well as transcription proteins. Epi-Decoder will enable the delineation of complex and dynamic protein-DNA interactions across many regions of the genome.

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Section: Discussionmentioning

confidence: 99%

Decoding the chromatin proteome of a single genomic locus by DNA sequencing

Korthout

Poramba‐Liyanage

Verzijlbergen

et al. 2017

Preprint

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“…The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system has facilitated widely used genome manipulation capabilities including targeted gene disruption 1,2 , transcriptional activation and repression 3 , epigenetic modification 3 , and direct conversion of a target base pair to a different base pair 4,5 in a broad range of organisms and cell types 6 . CRISPR-Cas9 targets DNA in a manner that is programmed by an RNA (typically a single-guide RNA, or sgRNA 7 ) that contains a “spacer” sequence complementary to the target DNA site, the “protospacer”.…”

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confidence: 99%

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity

Miller

Geurts

et al. 2018

Nature

1,286

1,094

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Summary A key limitation to the use of CRISPR-Cas9 proteins for genome editing and other applications is the requirement that a protospacer adjacent motif (PAM) be present at the target site. For the most commonly used Cas9 from Streptococcus pyogenes (SpCas9), this PAM requirement is NGG. No natural or engineered Cas9 variants shown to function efficiently in mammalian cells offer a PAM less restrictive than NGG. Here we used phage-assisted continuous evolution (PACE) to evolve an expanded PAM SpCas9 variant (xCas9) that can recognize a broad range of PAM sequences including NG, GAA, and GAT. The PAM compatibility of xCas9 is the broadest reported to date among Cas9s active in mammalian cells, and supports applications in human cells including targeted transcriptional activation, nuclease-mediated gene disruption, and both cytidine and adenine base editing. Remarkably, despite its broadened PAM compatibility, xCas9 has much greater DNA specificity than SpCas9, with substantially lower genome-wide off-target activity at all NGG target sites tested, as well as minimal off-target activity when targeting genomic sites with non-NGG PAMs. These findings expand the DNA targeting scope of CRISPR systems and establish that there is no necessary trade-off between Cas9 editing efficiency, PAM compatibility, and DNA specificity.

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“…Therefore, the CRISPR/Cas9 system can only be applied to sequences with proximal to the PAM motifs. A solution is a discovery or engineering of new Cas nucleases that require motifs other than -NGG-PAM (Mitsunobu, Teramoto, Nishida, & Kondo, 2017;Schwartz et al, 2016). Another disadvantage of the CRISPR/Cas9 system is an off-site effect, which occurs when the nucleotides that drive the CRISPR/Cas9 complex recognize target sequences with mismatched bases, generating undesirable multiple DSBs (Hsu et al, 2013).…”

Section: The Authors Tested Various Codon-optimizedmentioning

confidence: 99%

History of genome editing in yeast

Fraczek

Naseeb

Delneri

2018

Yeast

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For thousands of years humans have used the budding yeast Saccharomyces cerevisiae for the production of bread and alcohol; however, in the last 30–40 years our understanding of the yeast biology has dramatically increased, enabling us to modify its genome. Although S. cerevisiae has been the main focus of many research groups, other non‐conventional yeasts have also been studied and exploited for biotechnological purposes. Our experiments and knowledge have evolved from recombination to high‐throughput PCR‐based transformations to highly accurate CRISPR methods in order to alter yeast traits for either research or industrial purposes. Since the release of the genome sequence of S. cerevisiae in 1996, the precise and targeted genome editing has increased significantly. In this ‘Budding topic’ we discuss the significant developments of genome editing in yeast, mainly focusing on Cre‐loxP mediated recombination, delitto perfetto and CRISPR/Cas.

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Beyond Native Cas9: Manipulating Genomic Information and Function

Cited by 64 publications

References 88 publications

Decoding the chromatin proteome of a single genomic locus by DNA sequencing

Decoding the chromatin proteome of a single genomic locus by DNA sequencing

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity

History of genome editing in yeast

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