Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria meningitidis

Rousseau, Beth A.; Hou, Zhonggang; Gramelspacher, Max J.; Zhang, Yan

doi:10.1016/j.molcel.2018.01.025

Cited by 78 publications

(69 citation statements)

References 48 publications

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“…It was shown that this activity is crRNA-, and tracrRNA-dependent, and that a site specific RNA cleavage is most probably performed by the Cas9 s HNH domain [58]. Neither PAM nor PFS sequences were shown to be required to regulate this activity [59]. To date, this particular ssRNA targeting activity has been described for Staphylococcus aureus [60], Campylobacter jejuni [58], and Neisseria meningitides [59].…”

Section: Type II (Cas9) Systemsmentioning

confidence: 99%

“…Neither PAM nor PFS sequences were shown to be required to regulate this activity [59]. To date, this particular ssRNA targeting activity has been described for Staphylococcus aureus [60], Campylobacter jejuni [58], and Neisseria meningitides [59]. The exact role of the ssRNA targeting activity of type II CRISPR-Cas systems remains unknown.…”

Section: Type II (Cas9) Systemsmentioning

confidence: 99%

“…Many variants of Cas9 target RNA naturally, without need of a PAM sequence [57][58][59][60]. Some of them were also shown to be programmable by alteration of the spacer sequences [58,59]. These Cas9 variants look very promising candidates for potential applications.…”

Section: Type Ii(cas9)-based Applicationsmentioning

confidence: 99%

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RNA-Targeting CRISPR–Cas Systems and Their Applications

Burmistrz

Krakowski

Krawczyk‐Balska

2020

IJMS

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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–CRISPR-associated (Cas) systems have revolutionized modern molecular biology. Numerous types of these systems have been discovered to date. Many CRISPR–Cas systems have been used as a backbone for the development of potent research tools, with Cas9 being the most widespread. While most of the utilized systems are DNA-targeting, recently more and more attention is being gained by those that target RNA. Their ability to specifically recognize a given RNA sequence in an easily programmable way makes them ideal candidates for developing new research tools. In this review we summarize current knowledge on CRISPR–Cas systems which have been shown to target RNA molecules, that is type III (Csm/Cmr), type VI (Cas13), and type II (Cas9). We also present a list of available technologies based on these systems.

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Section: Type II (Cas9) Systemsmentioning

confidence: 99%

Section: Type II (Cas9) Systemsmentioning

confidence: 99%

See 1 more Smart Citation

RNA-Targeting CRISPR–Cas Systems and Their Applications

Burmistrz

Krakowski

Krawczyk‐Balska

2020

IJMS

View full text Add to dashboard Cite

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“…Some enzymes simply fail to work and some choose their substrates promiscuously, necessitating thorough biochemical characterization [59][60][61][62][63][64][65] . Nevertheless, orthologous Cas9 enzymes with different PAM requirements increase targeting flexibility and allow multiplexing for combinatorial genetic screens, as demonstrated for SpCas9 and SaCas9 6,11,66 .…”

Section: Article Preprintmentioning

confidence: 99%

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Agudelo

Carter

Velimirovic

et al. 2018

Preprint

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The broad spectrum of activities displayed by CRISPR-Cas systems has led to biotechnological innovations that are poised to transform human therapeutics. Therefore, the comprehensive characterization of distinct Cas proteins is highly desirable. Here we expand the repertoire of nucleases for mammalian genome editing using the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9). We define functional protospacer adjacent motif (PAM) sequences and variables required for robust and efficient editing in vitro. Expression of holoSt1Cas9 from a single adeno-associated viral (rAAV) vector in the neonatal liver rescued lethality and metabolic defects in a mouse model of hereditary tyrosinemia type I demonstrating effective cleavage activity in vivo. Furthermore, we identified potent anti-CRISPR proteins to regulate the activity of both St1Cas9 and the related type II-A Staphylococcus aureus Cas9 (SaCas9). This work expands the targeting range and versatility of CRISPR-associated enzymes and should encourage studies to determine its structure, genome-wide specificity profile and sgRNA design rules. INTRODUCTIONClustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins form a prokaryotic adaptive immune system and some of its components have been harnessed for robust genome editing 1 . Type II-based editing tools rely on a large multidomain endonuclease, Cas9, guided to its DNA target by an engineered single-guide RNA (sgRNA) chimera 2 (See 3, 4 for a classification of CRISPR-Cas systems). The Cas9-sgRNA binary complex finds its target through recognition of a short sequence called the protospacer adjacent motif (PAM) and subsequent base pairing of the guide RNA with the DNA to generate a specific doublestrand break (DSB) 1,5 . While Streptococcus pyogenes (SpCas9) remains the most widely used Cas9 variant for genome engineering, the diversity of naturally occurring RNA-guided nucleases is astonishing 4 . Hence, Cas9 enzymes from different microbial species can contribute to the expansion of the CRISPR toolset by increasing targeting density, improving activity and specificity as well as easing delivery 1,6 .In principle, engineering complementary CRISPRCas systems from distinct bacterial species should be relatively straightforward, as they have been minimized to only two components. However, many such enzymes were found inactive in human cells despite being accurately reprogrammed for DNA binding and cleavage in vitro [7][8][9][10] . Nevertheless, the full potential of selected enzymes can be unleashed using machine learning to establish sgRNA design rules 11,12 .Perhaps the most striking example of the value of alternative Cas9 enzymes is the implementation of the type II-A Cas9 from Staphylococcus aureus (SaCas9) for in vivo editing using recombinant adeno-associated virus (rAAV) vectors 7,13, 14 . More recently, Campylobacter jejuni and Neisseria meningitidis Cas9s from the type II-C 15 CRISPRCas systems have been added to this repertoire 16,17 .In vivo genome edi...

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“…Targeting of RCas9 to repetitive sequences does not require PAMmer 23 , however repeat sequences constitute only a small proportion of all RNA cis-regulatory elements. Following the initial report of RCas9, other CRISPR/Cas9 systems were also found to bind to single-stranded RNA in vitro 24,25 , but evidence for their in vivo RNA binding in mammalian cells is lacking. RNA-guided RNA nucleases from bacterial CRISPR systems has recently been discovered [26][27][28] .…”

mentioning

confidence: 99%

CRISPR Artificial Splicing Factors

Jillette

Cheng

2018

Preprint

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We report here the engineering of CRISPR Artificial Splicing Factors (CASFx) based on an RNA-targeting CRISPR/Cas system. We showed that simultaneous exon inclusion and exclusion can be induced at distinct targets by differential positioning of CASFx. We also created inducible CASFx (iCASFx) using the FKBP-FRB chemical-inducible dimerization domain, allowing small molecule control of alternative splicing. MainSplicing is a process in which segments of a pre-mRNA called introns are removed while segments called exons are joined together to form mature mRNA 1 . Alternative splicing is a phenomenon in which different exon segments are spliced together to form mature mRNA with different sequences, greatly expanding the protein repertoire by allowing different proteins to be coded by a single gene. The process of alternative splicing is deeply embedded in gene regulatory networks by controlling gene isoform expression of >90% of human genes 2 . Given such prevalence, dysregulation of (alternative) splicing has been implicated in many diseases 3-5 .RNA-seq is a powerful method to "read" transcriptomes for changes in alternative splicing in

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Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria meningitidis

Cited by 78 publications

References 48 publications

RNA-Targeting CRISPR–Cas Systems and Their Applications

RNA-Targeting CRISPR–Cas Systems and Their Applications

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

CRISPR Artificial Splicing Factors

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