Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality

Briner, Alexandra E.; Donohoue, Paul D.; Gomaa, Ahmed A.; Selle, Kurt; Slorach, Euan M.; Nye, Christopher H.; Haurwitz, R.E.; Beisel, Chase L.; May, Andrew P.; Barrangou, Rodolphe

doi:10.1016/j.molcel.2014.09.019

Cited by 233 publications

(269 citation statements)

References 26 publications

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“…Thus, a population targeting the protospacer/PAM : : plasmid would exhibit a lower transformation 'efficiency' than the pGK12 vector control due to plasmid cleavage. Indeed, consistent with other studies (Marraffini & Sontheimer, 2008;Gomaa et al, 2014), targeting of plasmids exhibiting perfect spacer : : PAM combinations manifested 3.13 and 3.89 log reductions in transformants of L. gasseri NCK 1342 and JV-V03, respectively (Fig. 5).…”

Section: R Sanozky-dawes and Otherssupporting

confidence: 91%

“…Characterization and exploitation of CRISPR-Cas systems in bacteria have led to effective typing and strain detection (Horvath et al, 2009;Shariat et al, 2013;, engineered immunity against mobile genetic elements (Garneau et al, 2010;, sequence-specific endogenous killing of bacteria (Gomaa et al, 2014) and programmable transcriptional regulation (Bikard et al, 2013;Luo et al, 2015). In silico surveys of lactic acid bacterial genomes suggest that these micro-organisms are a significant reservoir for orthogonal type II CRISPR-Cas systems (Chylinski et al, 2013; Briner…”

Section: Mechanisms Of Escape From the Crispr-cas Systemmentioning

confidence: 99%

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Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri

et al. 2015

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Bacteria encode clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated genes (cas), which collectively form an RNA-guided adaptive immune system against invasive genetic elements. In silico surveys have revealed that lactic acid bacteria harbour a prolific and diverse set of CRISPR-Cas systems. Thus, the natural evolutionary role of CRISPR-Cas systems may be investigated in these ecologically, industrially, scientifically and medically important microbes. In this study, 17 Lactobacillus gasseri strains were investigated and 6 harboured a type II-A CRISPR-Cas system, with considerable diversity in array size and spacer content. Several of the spacers showed similarity to phage and plasmid sequences, which are typical targets of CRISPR-Cas immune systems. Aligning the protospacers facilitated inference of the protospacer adjacent motif sequence, determined to be 59-NTAA-39 flanking the 39 end of the protospacer. The system in L. gasseri JV-V03 and NCK 1342 interfered with transforming plasmids containing sequences matching the most recently acquired CRISPR spacers in each strain. We report the distribution and function of a native type II-A CRISPR-Cas system in the commensal species L. gasseri. Collectively, these results open avenues for applications for bacteriophage protection and genome modification in L. gasseri, and contribute to the fundamental understanding of CRISPR-Cas systems in bacteria.

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Section: R Sanozky-dawes and Otherssupporting

confidence: 91%

Section: Mechanisms Of Escape From the Crispr-cas Systemmentioning

confidence: 99%

Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri

et al. 2015

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“…2A). The nexus motif, recently shown to be critical for activity (11), occupies a central position between the lobes and forms extensive interactions with the bridge helix. Based on this interaction profile, we generated a full-length sgRNA and two shorter sgRNA constructs that were selectively truncated from either the 5′ or 3′ end ( Fig.…”

Section: Significancementioning

confidence: 99%

Rational design of a split-Cas9 enzyme complex

Wright

Sternberg²,

Taylor

et al. 2015

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

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Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.CRISPR-Cas9 | genome engineering | split enzyme B acteria use RNA-guided adaptive immune systems encoded by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) genomic loci to defend against invasive DNA (1, 2). In type II CRISPR-Cas systems, a single enzyme called Cas9 is responsible for targeting and cleavage of foreign DNA (3). The ability to program Cas9 for DNA cleavage at sites defined by engineered single-guide RNAs (sgRNAs) (4) has led to its adoption as a robust and versatile platform for genome engineering (for recent reviews, see refs. 5-7).Cas9 contains two nuclease active sites that function together to generate DNA double-strand breaks (DSBs) at sites complementary to the 20-nt guide RNA sequence and adjacent to a protospacer adjacent motif (PAM). Structural studies of the Streptococcus pyogenes Cas9 showed that the protein exhibits a bilobed architecture comprising the catalytic nuclease lobe and the α-helical lobe of the enzyme (8). Electron microscopy (EM) studies and comparisons with X-ray crystal structures with and without a bound guide RNA and target DNA revealed a largescale conformational rearrangement of the two lobes relative to each other upon nucleic acid binding (8, 9). Strikingly, RNA binding induces the nuclease lobe to rotate ∼100°relative to the α-helical lobe, generating a nucleic-acid binding cleft that can accommodate DNA, and interactions between the two lobes seem to be mediated primarily through contacts with the bound nucleic acid rather than direct protein-protein contacts (8, 9). These observations suggested that the two structural lobes of Cas9 might be separable into independent polypeptides that retain the ability to assemble into an active enzyme complex. Such a system would enable analysis of the functionally distinct properties of each Cas9 structural region and might offer a unique mechanism for control...

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“…Accordingly, genomic analysis of S. thermophilus and its bacteriophages established a likely mechanism for phage/DNA protection in CRISPR-Cas systems. Investigation of CRISPR-Cas systems in S. thermophilus led to bioinformatic analysis of spacer origin (4,20), discovery of the proto-spacer adjacent motif (PAM) sequences (19,21), understanding of phage-host dynamics (22,23), demonstration of Cas9 endonuclease activity (7,24,25), and, recently, determination of the transactivating crRNA tracrRNA structural motifs governing function and orthogonality of type II systems (26). Genomic analysis of S. thermophilus revealed evolutionary…”

mentioning

confidence: 99%

CRISPR-based screening of genomic island excision events in bacteria

Selle

Klaenhammer

Barrangou

2015

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

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Genomic analysis of Streptococcus thermophilus revealed that mobile genetic elements (MGEs) likely contributed to gene acquisition and loss during evolutionary adaptation to milk. Clustered regularly interspaced short palindromic repeats-CRISPR-associated genes (CRISPR-Cas), the adaptive immune system in bacteria, limits genetic diversity by targeting MGEs including bacteriophages, transposons, and plasmids. CRISPR-Cas systems are widespread in streptococci, suggesting that the interplay between CRISPRCas systems and MGEs is one of the driving forces governing genome homeostasis in this genus. To investigate the genetic outcomes resulting from CRISPR-Cas targeting of integrated MGEs, in silico prediction revealed four genomic islands without essential genes in lengths from 8 to 102 kbp, totaling 7% of the genome. In this study, the endogenous CRISPR3 type II system was programmed to target the four islands independently through plasmid-based expression of engineered CRISPR arrays. Targeting lacZ within the largest 102-kbp genomic island was lethal to wild-type cells and resulted in a reduction of up to 2.5-log in the surviving population. Genotyping of Lac − survivors revealed variable deletion events between the flanking insertion-sequence elements, all resulting in elimination of the Lac-encoding island. Chimeric insertion sequence footprints were observed at the deletion junctions after targeting all of the four genomic islands, suggesting a common mechanism of deletion via recombination between flanking insertion sequences. These results established that self-targeting CRISPR-Cas systems may direct significant evolution of bacterial genomes on a population level, influencing genome homeostasis and remodeling.CRISPR | lactic acid bacteria | transposons | IS-elements | Cas9

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Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality

Cited by 233 publications

References 26 publications

Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri

Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri

Rational design of a split-Cas9 enzyme complex

CRISPR-based screening of genomic island excision events in bacteria

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