In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni

Kim, Eunji; Koo, Taeyoung; Park, Sung Wook; Kim, Daesik; Kim, Kyoungmi; Cho, Hee Yeon; Song, Dong Woo; Lee, Kyu Jun; Jung, Mi; Kim, Seokjoong; Kim, Jin Hyoung; Kim, Jeong Hun; Kim, Jin-Soo

doi:10.1038/ncomms14500

Cited by 581 publications

(527 citation statements)

References 35 publications

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“…Other natural CRISPR nucleases shown to function efficiently in mammalian cells include Staphylococcus aureus Cas9 (SaCas9) 10 , Acidaminococcus sp. Cpf1 11 , Lachnospiraceae bacterium Cpf1 11 , Campylobacter jejuni Cas9 12 , Streptococcus thermophilus Cas9 13 , and Neisseria meningitides Cas9 14 . None of these mammalian cell-compatible CRISPR nucleases, however, offer a PAM that occurs as frequently as that of SpCas9.…”

mentioning

confidence: 99%

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity

Miller

Geurts

et al. 2018

Nature

1,319

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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confidence: 99%

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity

Miller

Geurts

et al. 2018

Nature

1,319

1,094

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“…The two Type II-C Cas9 orthologs (NmeCas9 and CjeCas9) that have been validated for 683 mammalian genome editing and assessed for genome-wide specificity (Lee et al 2016;Kim et al 2017) 684…”

Section: Truncated Sgrnas Reduce Off-target Cleavage By Nmecas9 571mentioning

confidence: 99%

NmeCas9 is an intrinsically high-fidelity genome editing platform

Amrani¹,

Gao²,

Liu³

et al. 2017

Preprint

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BackgroundThe development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wild-type SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large (e.g. mammalian) genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large (~4.2 kb) open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs (e.g. from Staphylococcus aureus, Campylobacter jejuni, Geobacillus stearothermophilus and Neisseria meningitidis) are considerably smaller and therefore better suited for viral delivery.ResultsHere we show that wild-type NmeCas9, when programmed with guide sequences of natural length (24 nucleotides), exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5’-N4GATT-3’), for NmeCas9 genome editing in human cells.ConclusionsOur results show that NmeCas9 is a naturally high-fidelity genome editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.

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“…Once a nuclease is chosen, it is important to ensure that gRNA target sequences are designed using the optimal length for that nuclease. For example, 20 bp is typically used for SpCas9 (Hsu et al, 2013), 22 bp for CjCas9 (Kim et al, 2017b), and 21–23 bp for SaCas9 (Ran et al, 2015). …”

Section: Library Designmentioning

confidence: 99%

High-Throughput Approaches to Pinpoint Function within the Noncoding Genome

2017

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The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas nuclease system is a powerful tool for genome editing, and its simple programmability has enabled high-throughput genetic and epigenetic studies. These high-throughput approaches offer investigators a toolkit for functional interrogation of not only protein-coding genes but also noncoding DNA. Historically, noncoding DNA has lacked the detailed characterization that has been applied to protein-coding genes in large part because there has not been a robust set of methodologies for perturbing these regions. Although the majority of high-throughput CRISPR screens have focused on the coding genome to date, an increasing number of CRISPR screens targeting noncoding genomic regions continue to emerge. Here, we review high-throughput CRISPR-based approaches to uncover and understand functional elements within the noncoding genome and discuss practical aspects of noncoding library design and screen analysis.

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In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni

Cited by 581 publications

References 35 publications

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity

NmeCas9 is an intrinsically high-fidelity genome editing platform

High-Throughput Approaches to Pinpoint Function within the Noncoding Genome

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