Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome

Müller, Maximilian; Lee, Ciaran M.; Gasiūnas, Giedrius; Davis, Timothy; Cradick, Thomas J.; Šikšnys, Virginijus; Bao, Gang; Cathomen, Toni; Mussolino, Claudio

doi:10.1038/mt.2015.218

Cited by 218 publications

(141 citation statements)

References 56 publications

(94 reference statements)

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“…Although the Cas9 ortholog from Streptococcus pyogenes strain SF370 [SpyCas9, subtype II-A (Makarova et al, 2015)] is the most commonly used and the best understood, type II CRISPR-Cas systems from several other bacterial species have also been adapted for eukaryotic genome editing (Cong et al, 2013; Esvelt et al, 2013; Hirano et al, 2016; Hou et al, 2013; Lee et al, 2016; Muller et al, 2016; Ran et al, 2015). For example, Cas9 from Neisseria meningitidis (NmeCas9), which belongs to the CRISPR-Cas subtype II-C (Makarova et al, 2015), is an effective tool for human genome editing (Esvelt et al, 2013; Hou et al, 2013; Lee et al, 2016, Amrani et al, manuscript in preparation).…”

Section: Introductionmentioning

confidence: 99%

Naturally Occurring Off-Switches for CRISPR-Cas9

Pawluk

Amrani

Zhang

et al. 2016

Cell

366

409

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Summary CRISPR-Cas9 technology would be enhanced by the ability to inhibit Cas9 function spatially, temporally, or conditionally. Previously, we discovered small proteins encoded by bacteriophages that inhibit the CRISPR-Cas systems of their host bacteria. These “anti-CRISPRs” were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. We posited that nature would also yield Cas9 inhibitors in response to the evolutionary arms race between bacteriophages and their hosts. Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9), and can be used as potent inhibitors of genome editing by this system in human cells. These anti-CRISPR proteins now enable “off-switches” for CRISPR-Cas9 activity, and provide a genetically-encodable means to inhibit CRISPR-Cas9 genome editing in eukaryotes.

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Section: Introductionmentioning

confidence: 99%

Naturally Occurring Off-Switches for CRISPR-Cas9

Pawluk

Amrani

Zhang

et al. 2016

Cell

366

409

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“…Presumptive solutions to this problem include the use of bioinformatics tool for selection of sgRNAs (www.biootools.com), such as CRISPRoffinder and direct use of inducible and non-inducible CRISPR/Cas9 proteins (Müller et al, 2016;Slaymaker et al, 2016). These applications have had partial achievement due to the different sequences and genetic framework of each target DNA.…”

Section: Potential Challenges and Recent Advances In Crispr/cas Systemsmentioning

confidence: 99%

A Review of CRISPR-Based Genome Editing: Survival, Evolution and Challenges

Ahmad¹,

Ahmad²,

Asif³

et al. 2018

Current Issues in Molecular Biology

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Precise nucleic acid editing technologies have facilitated the research of cellular function and the development of novel therapeutics, especially the current programmable nucleases-based editing tools, such as the prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases (Cas). As CRISPR-based therapies are advancing toward human clinical trials, it is important to understand how natural genetic variation in the human population may affect the results of these trials and even patient safety. The development of "base-editing" technique allows the direct, stable transformation of target DNA base into an alternative in a programmable way, without DNA double strand cleavage or a donor template. Genome-editing techniques hold promises for the treatment of genetic disease at the DNA level by blocking the sequences associated with disease from producing disease-causing proteins. Currently, scientists can select the gene they want to modify, use the Cas9 as a "molecular cutter" to cut it out, and transform it into a more desirable version. In this review, we focus on the recent advances of CRISPR/Cas system by outlining the evolutionary and biotechnological implications of current strategies for improving the specificity and accuracy of these genome-editing technologies.

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“…This restricts the range of accessible targets but may reduce the occurrence of offtarget mutagenesis 73 . The consensus PAM for St1Cas9 (LMD-9 and DGCC7710 S. thermophilus strains that differ by only 2 aa) has been defined as N 1 N 2 A 3 G 4 A 5 A 6 (W 7 ), however sequences closely related to the consensus can be functional in test tubes and in bacterial cells 27,35,[74][75][76][77] .…”

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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Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome

Cited by 218 publications

References 56 publications

Naturally Occurring Off-Switches for CRISPR-Cas9

Naturally Occurring Off-Switches for CRISPR-Cas9

A Review of CRISPR-Based Genome Editing: Survival, Evolution and Challenges

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

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