A Programmable Dual-rna-guided Dna Endonuclease in Adaptive Bacterial Immunity Review
Science. Author manuscript; available in PMC 2018 Dec 7.
Published in final edited class as:
PMCID: PMC6286148
HHMIMSID: HHMIMS995853
A programmable dual RNA-guided Deoxyribonucleic acid endonuclease in adaptive bacterial immunity
Martin Jinek
1Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA.
2Department of Molecular and Cell Biology, Academy of California, Berkeley, California 94720, USA.
Krzysztof Chylinski
3Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria.
4The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Inquiry (UCMR), Department of Molecular Biology, Umeå University, Southward-90187 Umeå, Sweden.
Ines Fonfara
4The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Inquiry (UCMR), Department of Molecular Biology, Umeå University, S-90187 Umeå, Sweden.
Michael Hauer
2Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, United states of america.
5Present address: Friedrich Miescher Institute for Biomedical Inquiry, 4058 Basel, Switzerland.
Jennifer A. Doudna
1Howard Hughes Medical Institute, Academy of California, Berkeley, California 94720, Us.
2Department of Molecular and Cell Biological science, University of California, Berkeley, California 94720, U.s.a..
half dozenDepartment of Chemical science, Academy of California, Berkeley, California 94720, USA.
7Physical Biosciences Sectionalisation, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Emmanuelle Charpentier
4The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Research (UCMR), Department of Molecular Biology, Umeå Academy, South-90187 Umeå, Sweden.
Abstract
CRISPR/Cas systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using crRNAs to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA base-paired to trans-activating tracrRNA forms a two-RNA construction that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target Deoxyribonucleic acid. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand while the Cas9 RuvC-similar domain cleaves the not-complementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the organization for RNA-programmable genome editing.
I-Sentence Summary:
A two-RNA construction directs an endonuclease to carve target DNA.
Leaner and archaea take evolved RNA-mediated adaptive defence systems called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-every bitsociated) that protect organisms from invading viruses and plasmids(1-3). These defense systems rely on small RNAs for sequence-specific detection and silencing of foreign nucleic acids. CRISPR/Cas systems are equanimous of cas genes organized in operon(south) and a CRISPR array consisting of unique genome-targeting sequences (chosen spacers) interspersed with identical repeats (1-three). CRISPR/Cas mediated immunity occurs in three steps. In the adaptive phase, leaner and archaea harboring one or more CRISPR loci respond to viral and plasmid challenge by integrating short fragments of strange sequence (protospacers) into the host chromosome at the proximal end of the CRISPR array (one-3). In the expression and interference phases, transcription of the repeat-spacer element into precursor CRISPR RNA (pre-crRNA) molecules followed by enzymatic cleavage yields the short crRNAs that tin base pair with complementary protospacer sequences of invading viral or plasmid targets (4-11). Target recognition by crRNAs directs the silencing of the foreign sequences by ways of Cas proteins that function in circuitous with the crRNAs (x, 12-20).
In that location are three types of CRISPR/Cas systems (21-23). The Blazon I and Iii systems share some overarching features: specialized Cas endonucleases process the pre-crRNAs, and once mature, each crRNA assembles into a large multi-Cas protein circuitous capable of recognizing and cleaving nucleic acids complementary to the crRNA. In contrast, Type II systems process pre-crRNAs by a different mechanism in which a trans-activating crRNA (tracrRNA) complementary to the repeat sequences in pre-crRNA triggers processing by the double-stranded RNA-specific ribonuclease RNase Three in the presence of the Cas9 (formerly Csn1) protein (4, 24) (fig. S1). Cas9 is thought to be the sole protein responsible for crRNA-guided silencing of strange Dna (25-27).
We evidence here that in Type Ii systems, Cas9 proteins plant a family of enzymes that require a base-paired construction formed betwixt the activating tracrRNA and the targeting crRNA to carve target double-stranded (ds) Deoxyribonucleic acid. Site-specific cleavage occurs at locations determined past both base-pairing complementarity between the crRNA and the target protospacer DNA and a short motif (referred to as the protospacer adjacent motif, or PAM) juxtaposed to the complementary region in the target DNA. Our report further demonstrates that the Cas9 endonuclease family can be programmed with single RNA molecules to cleave specific DNA sites, thereby raising the exciting possibility of developing a simple and versatile RNA-directed organization to generate dsDNA breaks (DSBs) for genome targeting and editing.
Cas9 is a DNA endonuclease guided by two RNAs.
Cas9, the hallmark protein of Type Ii systems, has been hypothesized to be involved in both crRNA maturation and crRNA-guided DNA interference (fig. S1) (4, 25-27). Cas9 is involved in crRNA maturation (4), but its direct participation in target DNA destruction has not been investigated. To test whether and how Cas9 might exist capable of target DNA cleavage, nosotros used an overexpression system to purify Cas9 protein derived from the pathogen Streptococcus pyogenes (fig. S2) and tested its ability to cleave a plasmid DNA or an oligonucleotide duplex bearing a protospacer sequence complementary to a mature crRNA, and a bona fide PAM. We establish that mature crRNA alone was incapable of directing Cas9-catalyzed plasmid DNA cleavage (Fig. 1A and fig. S3A). All the same, add-on of tracrRNA, which can base pair with the repeat sequence of crRNA and is essential to crRNA maturation in this arrangement, triggered Cas9 to carve plasmid DNA (Fig. 1A and fig. S3A). The cleavage reaction required both magnesium and the presence of a crRNA sequence complementary to the Deoxyribonucleic acid; a crRNA capable of tracrRNA base-pairing but containing a not-cognate target Deoxyribonucleic acid-bounden sequence did not back up Cas9-catalyzed plasmid cleavage (Fig. 1A, fig. S3A, compare crRNA-sp2 to crRNA-sp1 and fig. S4A). Similar results were obtained with a short linear dsDNA substrate (Fig. 1B and fig. S3B, C). The trans-activating tracrRNA is thus a small not-coding RNA with two disquisitional functions: triggering pre-crRNA processing past the enzyme RNase III (4) and later activating crRNA-guided Deoxyribonucleic acid cleavage by Cas9.
Cas9 is a Deoxyribonucleic acid endonuclease guided by two RNA molecules.
(A) Cas9 was programmed with a 42-nucleotide crRNA-sp2 (crRNA containing spacer two sequence) in the presence or absence of 75-nucleotide tracrRNA. The circuitous was added to round or XhoI-linearized plasmid DNA bearing a sequence complementary to spacer 2 and a functional PAM. crRNA-sp1, specificity control; Yard, DNA marker; refer to fig. S3A. (B) Cas9 was programmed with crRNA-sp2 and tracrRNA (nucleotides 4-89). The complex was incubated with double- or unmarried-stranded DNAs harboring a sequence complementary to spacer 2 and a functional PAM (4). The complementary or non-complementary strands of the Dna were 5'-radiolabeled and annealed with a non-labeled partner strand. Refer to fig. S3B, C. (C) Sequencing assay of cleavage products from Fig. 1A. Termination of primer extension in the sequencing reaction indicates the position of the cleavage site. The 3' last A overhang (asterisk) is an artifact of the sequencing reaction. Refer to fig. S5A, C. (D) The cleavage products from Fig. 1B were analyzed aslope five' terminate-labeled size markers derived from the complementary and non-complementary strands of the target Deoxyribonucleic acid duplex. M, marker; P, cleavage product. Refer to fig. S5B, C. (E) Schematic representation of tracrRNA, crRNA-sp2 and protospacer 2 DNA sequences; regions of crRNA complementarity to tracrRNA (orange) and the protospacer Dna (yellowish) are represented; PAM sequence, grey; cleavage sites mapped in (C) and (D) are represented by blue arrows (C), a red arrow (D, complementary strand) and a red line (D, not-complementary strand).
Cleavage of both plasmid and brusk linear dsDNA by tracrRNA:crRNA-guided Cas9 is site-specific (Fig. 1C-Due east and fig. S5A, B). Plasmid Dna cleavage produced edgeless ends at a position three base pairs upstream of the PAM sequence (Fig. 1C, E and fig. S5A, C) (26). Similarly, within short dsDNA duplexes, the Dna strand that is complementary to the target-binding sequence in the crRNA (the complementary strand) is cleaved at a site 3 base pairs upstream of the PAM (Fig. 1D, E and fig. S5B, C). The non-complementary Deoxyribonucleic acid strand is cleaved at one or more sites inside 3 to 8 base of operations pairs upstream of the PAM. Further investigation revealed that the non-complementary strand is first cleaved endonucleolytically and subsequently trimmed by a 3'-5' exonuclease activity (fig. S4B). The cleavage rates past Cas9 under single turnover atmospheric condition ranged from 0.iii to ane min−1, comparable to those of restriction endonucleases (fig. S6A), while incubation of wild-type Cas9-tracrRNA:crRNA circuitous with a 5-fold tooth backlog of substrate DNA provided evidence that the dual-RNA-guided Cas9 is a multiple-turnover enzyme (fig. S6B). In contrast to the CRISPR Type I Cascade complex (20), Cas9 cleaves both linearized and supercoiled plasmids (Fig. 1A, 2A). An invading plasmid can therefore in principle be cleaved multiple times past Cas9 proteins programmed with different crRNAs.
Cas9 uses two nuclease domains to cleave the two strands in the target DNA.
(A) Top: Schematic representation of Cas9 domain construction showing the positions of domain mutations. Bottom: Complexes of wild-type or nuclease mutant Cas9 proteins with tracrRNA:crRNA-sp2 were assayed for endonuclease activeness as in Fig. 1A. (B) Complexes of wild-type Cas9 or nuclease domain mutants with tracrRNA and crRNA-sp2 were tested for activity as in Fig. 1B.
Each Cas9 nuclease domain cleaves ane Deoxyribonucleic acid strand.
Cas9 contains domains homologous to both HNH and RuvC endonucleases (Fig. 2A, fig. S7) (21-23, 27, 28). We designed and purified Cas9 variants containing inactivating point mutations in the catalytic residues of either the HNH or RuvC-like domains (Fig. 2A and fig. S7) (23, 27). Incubation of these variant Cas9 proteins with native plasmid DNA showed that dual-RNA-guided mutant Cas9 proteins yielded nicked open round plasmids, while the wild-type Cas9 protein-tracrRNA:crRNA circuitous produced a linear Deoxyribonucleic acid product (Fig. 1A, 2A and fig. S3A, S8A). This effect indicates that the Cas9 HNH and RuvC-like domains each cleave one plasmid Dna strand. To decide which strand of the target Deoxyribonucleic acid is broken by each Cas9 catalytic domain, we incubated the mutant Cas9-tracrRNA:crRNA complexes with short dsDNA substrates in which either the complementary or the non-complementary strand was radiolabeled at its 5' stop. The resulting cleavage products indicated that the Cas9 HNH domain cleaves the complementary Dna strand, while the Cas9 RuvC-similar domain cleaves the not-complementary DNA strand (Fig. 2B and fig. S8B).
Dual-RNA requirements for target Deoxyribonucleic acid bounden and cleavage.
tracrRNA might be required for target DNA binding and/or to stimulate the nuclease activity of Cas9 downstream of target recognition. To distinguish betwixt these possibilities, we used an electrophoretic mobility shift analysis to monitor target Deoxyribonucleic acid bounden past catalytically inactive Cas9 in the presence or absence of crRNA and/or tracrRNA. Addition of tracrRNA substantially enhanced target Deoxyribonucleic acid binding by Cas9, whereas piffling specific DNA binding was observed with Cas9 solitary or Cas9-crRNA (fig. S9). This indicates that tracrRNA is required for target Dna recognition, possibly by properly orienting the crRNA for interaction with the complementary strand of target DNA. The predicted tracrRNA:crRNA secondary structure includes base-pairing between the iii'-terminal 22-nucleotides of the crRNA and a segment near the 5' end of the mature tracrRNA (Fig. 1E). This interaction creates a construction in which the 5'-last 20 nucleotides of the crRNA, which vary in sequence in different crRNAs, are available for target Deoxyribonucleic acid binding. The bulk of the tracrRNA downstream of the crRNA base-pairing region is free to form additional RNA structure(s) and/or to interact with Cas9 or the target Dna site. To determine whether the unabridged length of the tracrRNA is necessary for site-specific Cas9-catalyzed DNA cleavage, we tested Cas9-tracrRNA:crRNA complexes reconstituted using full-length mature (42-nt) crRNA and various truncated forms of tracrRNA lacking sequences at their five' or 3' ends. These complexes were tested for cleavage using a brusk target dsDNA. A substantially truncated version of the tracrRNA retaining nucleotides 23-48 of the native sequence was capable of supporting robust dual-RNA-guided Cas9-catalyzed Deoxyribonucleic acid cleavage (Fig. 3A, C and fig. S10A, B). Truncation of the crRNA from either terminate showed that Cas9-catalyzed cleavage in the presence of tracrRNA could be triggered with crRNAs missing the 3'-terminal ten nucleotides (Fig. 3B, C). In contrast, a 10-nucleotide deletion from the 5' stop of crRNA abolished Dna cleavage past Cas9 (Fig. 3B). We also analyzed Cas9 orthologs from diverse bacterial species for their ability to support S. pyogenes tracrRNA:crRNA-guided DNA cleavage. In contrast to closely related South. pyogenes Cas9 orthologs, more distantly related orthologs were not functional in the cleavage reaction (fig. S11). Similarly, S. pyogenes Cas9 guided by tracrRNA:crRNA pairs originating from more distant systems were unable to cleave DNA efficiently (fig. S11). Species specificity of dual-RNA-guided cleavage of DNA indicates co-evolution of Cas9, tracrRNA and the crRNA repeat, as well as the existence of a still unknown structure and/or sequence in the dual-RNA that is critical for the formation of the ternary complex with specific Cas9 orthologs.
Cas9-catalyzed cleavage of target DNA requires an activating domain in tracrRNA and is governed by a seed sequence in the crRNA.
(A) Cas9-tracrRNA:crRNA complexes were reconstituted using 42-nucleotide crRNA-sp2 and truncated tracrRNA constructs and assayed for cleavage action as in Fig. 1B. (B) Cas9 programmed with full-length tracrRNA and crRNA-sp2 truncations was assayed for activity as in (A). (C) Minimal regions of tracrRNA and crRNA capable of guiding Cas9-mediated Deoxyribonucleic acid cleavage (blue box). (D) Plasmids containing wild-type or mutant protospacer 2 sequences with indicated bespeak mutations (right) were cleaved in vitro by programmed Cas9 as in Fig. 1A (left top) and used for transformation assays of wild-type or pre-crRNA-deficient S. pyogenes (left bottom). The transformation efficiency was calculated as CFU per μg of plasmid Deoxyribonucleic acid; error bars represent standard deviations for 3 biological replicates. (E) Plasmids containing wild-blazon and mutant protospacer two inserts with varying extent of crRNA-target Dna mismatches (right) were cleaved in vitro by programmed Cas9 (left). The cleavage reactions were further digested with XmnI. The 1880 bp and 800 bp fragments are Cas9-generated cleavage products.
To investigate the protospacer sequence requirements for Type Ii CRISPR/Cas immunity in bacterial cells, a series of protospacer-containing plasmid DNAs harboring single-nucleotide mutations were analyzed for their maintenance post-obit transformation in Southward. pyogenes and their ability to be cleaved past Cas9 in vitro. In contrast to indicate mutations introduced at the v' end of the protospacer, mutations in the region shut to the PAM and the Cas9 cleavage sites were not tolerated in vivo and resulted in decreased plasmid cleavage efficiency in vitro (Fig. 3D). Our results are in understanding with a previous report of protospacer escape mutants selected in the Type Two CRISPR system from Due south. thermophilus in vivo (27, 29). Furthermore, the plasmid maintenance and cleavage results hint at the being of a "seed" region located at the three' end of the protospacer sequence that is crucial for the interaction with crRNA and subsequent cleavage past Cas9. In support of this notion, Cas9 enhanced complementary Dna strand hybridization to the crRNA and this enhancement was the strongest in the 3'-concluding region of the crRNA targeting sequence (fig. S12). Corroborating this, a contiguous stretch of at least 13 base pairs between the crRNA and the target Deoxyribonucleic acid site proximal to the PAM is required for efficient target cleavage, while up to six contiguous mismatches in the 5'-terminal region of the protospacer are tolerated (Fig. 3E). These findings are reminiscent of the previously observed seed sequence requirements for target nucleic acid recognition in Argonaute proteins (thirty, 31) and the Cascade and Csy CRISPR complexes (xiii, 14).
A brusque sequence motif dictates R-loop formation.
In multiple CRISPR/Cas systems, recognition of self versus non-self has been shown to involve a short sequence motif that is preserved in the foreign genome, referred to equally the PAM (27, 29, 32-34). PAM motifs are only a few base pairs in length, and their precise sequence and position vary co-ordinate to the CRISPR/Cas arrangement blazon (32). In the S. pyogenes Blazon Two system, the PAM conforms to an NGG consensus sequence, containing ii Chiliad:C base pairs that occur one base pair downstream of the crRNA binding sequence, inside the target DNA (4). Transformation assays demonstrated that the GG motif is essential for protospacer plasmid DNA elimination by CRISPR/Cas in bacterial cells (fig. S13A), consistent with previous observations in S. thermophilus (27). The motif is also essential for in vitro protospacer plasmid cleavage by tracrRNA:crRNA-guided Cas9 (fig. S13B). To decide the function of the PAM in target Dna cleavage by the Cas9-tracrRNA:crRNA complex, we tested a serial of dsDNA duplexes containing mutations in the PAM sequence on the complementary or non-complementary strands, or both (Fig. 4A). Cleavage assays using these substrates showed that Cas9-catalyzed DNA cleavage was specially sensitive to mutations in the PAM sequence on the not-complementary strand of the Deoxyribonucleic acid, in contrast to complementary strand PAM recognition past Type I CRISPR/Cas systems (20, 34). Cleavage of target single-stranded DNAs was unaffected by mutations of the PAM motif. This observation suggests that the PAM motif is required only in the context of target dsDNA and may thus be required to license duplex unwinding, strand invasion, and the formation of an R-loop structure. Using a dissimilar crRNA-target Deoxyribonucleic acid pair (crRNA-sp4 and protospacer iv DNA), selected due to the presence of a canonical PAM not present in the protospacer 2 target Dna, we institute that both M nucleotides of the PAM were required for efficient Cas9-catalyzed Dna cleavage (Fig. 4B and fig. S13C). To make up one's mind whether the PAM plays a straight part in recruiting the Cas9-tracrRNA:crRNA circuitous to the correct target DNA site, bounden affinities of the complex for target Dna sequences were analyzed past native gel mobility shift assays (Fig. 4C). Mutation of either G in the PAM sequence substantially reduced the analogousness of Cas9-tracrRNA:crRNA for the target DNA. This finding argues for specific recognition of the PAM sequence past Cas9 as a prerequisite for target Deoxyribonucleic acid binding and possibly strand separation to permit strand invasion and R-loop formation, which would exist analogous to the PAM sequence recognition by CasA/Cse1 implicated in a Type I CRISPR/Cas arrangement (34).
A PAM is required to license target DNA cleavage by the Cas9-tracrRNA:crRNA complex.
(A) Dual RNA-programmed Cas9 was tested for activeness as in Fig. 1B. Wild-type and mutant PAM sequences in target DNAs are indicated (right). (B) Protospacer 4 target DNA duplexes (labeled at both five' ends) containing wild-type and mutant PAM motifs were incubated with Cas9 programmed with tracrRNA (nt 23-89):crRNA-sp4. At indicated time points (min), aliquots of the cleavage reaction were taken and analyzed as in Fig. 1B. (C) Electrophoretic mobility shift assays were performed using RNA-programmed Cas9 (D10A/H840A) and protospacer 4 target DNA duplexes (same equally in (B)) containing wild-type and mutated PAM motifs. Cas9 (D10A/H840A)-RNA complex was titrated from 100 pM to 1 μM.
Cas9 tin exist programmed with a unmarried chimeric RNA.
Examination of the probable secondary structure of the tracrRNA:crRNA duplex (Fig. 1E, 3C) suggested the possibility that the features required for site-specific Cas9-catalyzed DNA cleavage could be captured in a single chimeric RNA. Although the tracrRNA:crRNA target selection machinery works efficiently in nature, the possibility of a single RNA-guided Cas9 is appealing due to its potential utility for programmed DNA cleavage and genome editing (Fig. 5A). We designed ii versions of a chimeric RNA containing a target recognition sequence at the 5' cease followed by a hairpin structure retaining the base-pairing interactions that occur betwixt the tracrRNA and the crRNA (Fig. 5B). This single transcript effectively fuses the 3'end of crRNA to the five' end of tracrRNA, thereby mimicking the dual-RNA structure required to guide site-specific Deoxyribonucleic acid cleavage by Cas9. In cleavage assays using plasmid Dna, we observed that the longer chimeric RNA was able to guide Cas9-catalyzed DNA cleavage in a fashion like to that observed for the truncated tracrRNA:crRNA duplex (Fig. 5B and fig. S14A). The shorter chimeric RNA did not work efficiently in this assay, confirming that nucleotides v-12 positions beyond the tracrRNA:crRNA base of operations-pairing interaction are important for efficient Cas9 binding and/or target recognition. Like results were observed in cleavage assays using curt dsDNA every bit a substrate, which further indicate that the position of the cleavage site in target DNA is identical to that observed using the dual tracrRNA:crRNA as a guide (Fig. 5C and fig. S14B). Finally, to institute whether the design of chimeric RNA might be universally applicative, we engineered five different chimeric guide RNAs to target a portion of the cistron encoding the green-fluorescent protein (GFP) (fig. S15A-C), and tested their efficacy against a plasmid carrying the GFP coding sequence in vitro. In all v cases, Cas9 programmed with these chimeric RNAs efficiently cleaved the plasmid at the right target site (Fig. 5D, fig. S15D), indicating that rational design of chimeric RNAs is robust and could in principle enable targeting of any Dna sequence of interest with few constraints beyond the presence of a GG dinucleotide adjacent to the targeted sequence.
Cas9 tin be programmed using a single engineered RNA molecule combining tracrRNA and crRNA features.
(A) Peak: In Blazon II CRISPR/Cas systems, Cas9 is guided by a two-RNA construction formed by activating tracrRNA and targeting crRNA to cleave site-specifically target dsDNA (refer to fig. S1). Bottom: A chimeric RNA generated past fusing the 3' end of crRNA to the 5' end of tracrRNA. (B) A plasmid harboring protospacer 4 target sequence and a wild-type PAM was subjected to cleavage past Cas9 programmed with tracrRNA(4-89):crRNA-sp4 duplex or in vitro-transcribed chimeric RNAs constructed past joining the 3' terminate of crRNA to the 5' end of tracrRNA with a GAAA tetraloop. Cleavage reactions were analyzed by restriction mapping with XmnI. Sequences of chimeric RNAs A and B are shown with DNA-targeting (yellow), crRNA repeat-derived (orangish) and tracrRNA-derived (calorie-free blue) sequences. (C) Protospacer 4 Deoxyribonucleic acid duplex cleavage reactions were performed every bit in Fig. 1B. (D) Five chimeric RNAs designed to target the GFP cistron were used to program Cas9 to carve a GFP gene-containing plasmid. Plasmid cleavage reactions were performed as in Fig. 3E, except that the plasmid Deoxyribonucleic acid was brake mapped with AvrII following Cas9 cleavage.
Conclusions.
In summary, nosotros place a Dna interference mechanism involving a dual-RNA structure that directs a Cas9 endonuclease to introduce site-specific double-stranded breaks in target Deoxyribonucleic acid. The tracrRNA:crRNA-guided Cas9 protein utilizes singled-out endonuclease domains, HNH and RuvC-like, to cleave the two strands in the target Deoxyribonucleic acid. Target recognition by Cas9 requires both a seed sequence in the crRNA and a GG dinucleotide-containing PAM sequence adjacent to the crRNA-binding region in the Deoxyribonucleic acid target. Nosotros further show that the Cas9 endonuclease can be programmed with guide RNA engineered as a single transcript to target and cleave any dsDNA sequence of involvement. The system is efficient, versatile and programmable past changing the DNA target-bounden sequence in the guide chimeric RNA. Zinc-Finger Nucleases (ZFNs) and Transcription-Activator Similar Effector Nucleases (TALENs) have attracted considerable involvement every bit artificial enzymes engineered to manipulate genomes (35-38). We propose an alternative methodology based on RNA-programmed Cas9 that could offer considerable potential for factor targeting and genome editing applications.
Supplementary Material
Supplement
Acknowledgements.
Nosotros thank Kaihong Zhou, Alison Marie Smith, Rachel Haurwitz and Sam Sternberg for excellent technical assist, and members of the Doudna and Charpentier laboratories and Jamie Cate for comments on the manuscript. We thank Barbara Meyer and Te-Wen Lo (UC Berkeley/HHMI) for providing the GFP plasmid. This work was funded by the Howard Hughes Medical Plant (M.J. and J.A.D.), the Austrian Scientific discipline Fund (W1207-B09, K.C. and Eastward.C.), the University of Vienna (K.C.), the Swedish Research Council (#K2010-57X-21436-01-3 and #621-2011-5752-LiMS, E.C.), the Kempe Foundation (E.C.) and Umeå University (K.C., Eastward.C.). J.A.D. is an Investigator and M.J. is a Inquiry Specialist of the Howard Hughes Medical Institute. G.C. is a fellow of the Austrian Doctoral Program in RNA Biology and co-supervised by R. Schroeder. We are grateful to A. Witte, U. Bläsi and R. Schroeder for helpful discussions, financial support to K.C and hosting M.C. in their laboratories at MFPL. Chiliad.J., K.C., J.A.D. and E.C. have filed a related patent.
Footnotes
Supplementary Materials. The Supplementary Materials incorporate the Supplementary Materials and Methods, Supplementary Figures S1-S15 with legends, and Supplementary Tables S1-S3, with references 39-47.
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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286148/
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