New CRISPR–Cas systems from uncultivated microbes

Burstein, David; Harrington, Lucas B.; Strutt, Steven; Probst, Alexander J.; Anantharaman, Karthik; Thomas, Brian C.; Doudna, Jennifer A.; Banfield, Jillian F.

doi:10.1038/nature21059

Cited by 507 publications

(397 citation statements)

References 68 publications

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“…Class 1 CRISPR-Cas systems are further divided into three types and 12 subtypes in all of which the effector modules are multisubunit complexes of Cas proteins (16). In contrast, in the currently identified three types and 12 subtypes of class 2, the effector modules are represented by a single multidomain protein, such as the thoroughly characterized Cas9 (15,17,18).…”

mentioning

confidence: 99%

Recruitment of CRISPR-Cas systems by Tn7-like transposons

Peters

Makarova

Shmakov

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

243

267

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A survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f, cas7f, and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates that evolution of the CRISPR-Cas-containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We show that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences and, in some cases, chromosomal sequences adjacent to the transposon. We hypothesize that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. These findings suggest the existence of RNAguided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element.CRISPR-Cas systems | Tn7 transposon | transposition strategy | crRNA guide | target-site selection

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mentioning

confidence: 99%

Recruitment of CRISPR-Cas systems by Tn7-like transposons

Peters

Makarova

Shmakov

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

243

267

View full text Add to dashboard Cite

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“…Nuclease deficient Cas9 has also been used for tagging genomic sites in wide-ranging applications [4][5][6] . This repurposing has revolutionized biology and sparked a search for other novel CRISPR-Cas enzymes 7,8 . One such search led to the discovery of the Cas protein Cpf1, with some of its orthologues reporting highly specific cleavage activities in mammalian cells [9][10][11][12] .…”

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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“…So far, only SpCas9 (1368 residues) has been tested for RNP-induced knock-in in plants. It could be interesting to test some of its analogs that are smaller in size, such as SaCas9 (1053 residues), StCas9 (1121), or NmCas9 (1082) [51], or the very recently discovered CasX (980) and CasY (1200) [97], to evaluate if the delivery of the corresponding RNP complexes to the nucleus would be facilitated.…”

Section: Transient Transformationmentioning

confidence: 99%

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

Collonnier

Guyon‐Debast

Maclot

et al. 2017

Methods

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a b s t r a c tBeyond its predominant role in human and animal therapy, the CRISPR-Cas9 system has also become an essential tool for plant research and plant breeding. Agronomic applications rely on the mastery of gene inactivation and gene modification. However, if the knock-out of genes by non-homologous end-joining (NHEJ)-mediated repair of the targeted double-strand breaks (DSBs) induced by the CRISPR-Cas9 system is rather well mastered, the knock-in of genes by homology-driven repair or end-joining remains difficult to perform efficiently in higher plants. In this review, we describe the different approaches that can be tested to improve the efficiency of CRISPR-induced gene modification in plants, which include the use of optimal transformation and regeneration protocols, the design of appropriate guide RNAs and donor templates and the choice of nucleases and means of delivery. We also present what can be done to orient DNA repair pathways in the target cells, and we show how the moss Physcomitrella patens can be used as a model plant to better understand what DNA repair mechanisms are involved, and how this knowledge could eventually be used to define more performant strategies of CRISPR-induced gene knock-in.

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New CRISPR–Cas systems from uncultivated microbes

Cited by 507 publications

References 68 publications

Recruitment of CRISPR-Cas systems by Tn7-like transposons

Recruitment of CRISPR-Cas systems by Tn7-like transposons

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

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

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