DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

doi:10.1038/nature13011

Cited by 1,672 publications

(1,859 citation statements)

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“…(AsCpf1), Lachnospiraceae bacterium (LbCpf1) and Francisella novicida (FnCpf1) 9 . Singlemolecule methods have been helpful in the study of CRISPR mechanisms [14][15][16][17][18][19][20][21][22][23] because they allow real-time detection of multiple and distinct steps of varying time lengths i.e. transient to longlived 24 .…”

mentioning

confidence: 99%

Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1

Singh

Mallon

Poddar

et al. 2017

Preprint

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CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We have used single-molecule fluorescence imaging and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologues. Like Cas9, Cpf1 initially binds DNA in search of PAM (protospacer-adjacent motif) sequences, verifies the target sequence unidirectionally from the PAM-proximal end and rapidly rejects any targets that lack a PAM or that are poorly matched with the guide-RNA. Cpf1 requires ~ 17 bp sequence match for both stable binding and cleavage, contrasting it with Cas9 which requires 9 bp for stable binding and ~16 bp for cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAMdistal cleavage product, but not the PAM-proximal product. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

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mentioning

confidence: 99%

Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1

Singh

Mallon

Poddar

et al. 2017

Preprint

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“…It is important to stress that CRISPR/Cas9 is a twostep process (Sternberg et al 2014, Jiang et al 2015: Cas9 only needs the annealing of 10 nt of the guide RNA to bind DNA, whereas its endonuclease activity requires the annealing of more than 16 nt to the genomic target (Kiani et al 2015). Direct microscopy study indicates that offtarget binding events are, on average, short-lived (Knight et al 2015).…”

Section: The Off-target Problemmentioning

confidence: 99%

CRISPR/Cas9: a breakthrough in generating mouse models for endocrinologists

Markossian¹,

Flamant²

2016

Journal of Molecular Endocrinology

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CRISPR/Cas9 is a recent development in genome editing which is becoming an indispensable element of the genetic toolbox in mice. It provides outstanding possibilities for targeted modification of the genome, and is often extremely efficient. There are currently two main limitations to in ovo genome editing in mice: the first is mosaicism, which is frequent in founder mice. The second is the difficulty to evaluate the advent of off-target mutations, which often imposes to wait for germline transmission to ensure genetic segregation between wanted and unwanted genetic mutations. However rapid progresses are made, suggesting that these difficulties can be overcome in the near future.

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“…4). For instance, the Cas9 nuclease involved in cutting might simultaneously repress 75 genes involved in NHEJ and therefore increase the frequency of HR 89 if supplied with a shortened guide RNA that directs it to bind and block transcription but not cut 90,91 . Alternatively, an orthogonal and nuclease-null Cas9 protein 92 encoded within the drive cassette could repress NHEJ genes and activate HR genes before activating the Cas9 nuclease.…”

Section: Copyingmentioning

confidence: 99%

Concerning RNA-Guided Gene Drives for the Alteration of Wild Populations

Esvelt

Smidler

Catteruccia

et al. 2014

Preprint

210

388

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Gene drives may be capable of addressing ecological problems by altering entire populations of wild organisms, but their use has remained largely theoretical due to technical constraints. Here we consider the potential for RNA-guided gene drives based on the CRISPR nuclease Cas9 to serve as a general method for spreading altered traits through wild populations over many generations. We detail likely capabilities, discuss limitations, and provide novel precautionary strategies to control the spread of gene drives and reverse genomic changes. The ability to edit populations of sexual species would offer substantial benefits to humanity and the environment. For example, RNA-guided gene drives could potentially prevent the spread of disease, support agriculture by reversing pesticide and herbicide resistance in insects and weeds, and control damaging invasive species. However, the possibility of unwanted ecological effects and near-certainty of spread across political borders demand careful assessment of each potential application. We call for thoughtful, inclusive, and well-informed public discussions to explore the responsible use of this currently theoretical technology.

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DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

Cited by 1,672 publications

References 48 publications

Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1

Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1

CRISPR/Cas9: a breakthrough in generating mouse models for endocrinologists

Concerning RNA-Guided Gene Drives for the Alteration of Wild Populations

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