The Experts below are selected from a list of 4737 Experts worldwide ranked by ideXlab platform
Kunihiko Naito - One of the best experts on this subject based on the ideXlab platform.
-
Generation of genetically modified mice using SpCas9-NG engineered nuclease
Scientific Reports, 2019Co-Authors: Wataru Fujii, Koji Sugiura, Arisa Ikeda, Haruka Ito, Takuya Kanke, Kunihiko NaitoAbstract:Although genetically modified mice can be generated with high efficiency by using CRISPR/Cas9-mediated genome editing in mouse zygotes, only the loci with a Protospacer-Adjacent Motif (PAM) sequence are targetable. The present study investigated the usability of engineered Streptococcus pyogenes Cas9 (SpCas9-NG) in mouse zygotes. In addition to the 5′-NGG sequence, SpCas9-NG recognized the 5′-NGA, 5′-NGC and 5′-NGT sequences in mouse zygotes as PAMs that were appropriate for the generation of knockout mice. Moreover, SpCas9-NG-mediated genome editing enabled the generation of knock-in mice untargetable by the conventional SpCas9 in mouse zygotes. These results suggest that SpCas9-NG-mediated genome editing in zygotes is available for the generation of knockout and knock-in mice at the locus corresponding to NGN-PAM.
-
Efficient Generation of Genome-Modified Mice Using Campylobacter jejuni-Derived CRISPR/Cas.
International journal of molecular sciences, 2017Co-Authors: Wataru Fujii, Koji Sugiura, Arisa Ikeda, Kunihiko NaitoAbstract:Mammalian zygote-mediated genome-engineering by Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas is currently used for the generation of genome-modified animals. Here, we report that a Campylobacter jejuni-derived orthologous CRISPR/Cas system recognizes a 5′-NNNVRYAC sequence as a Protospacer-Adjacent Motif in mouse zygotes, and is applicable for efficient generation of knockout mice. Moreover, this novel CRISPR/Cas can be used for zygote-mediated knock-in at a unique locus, suggesting that this system could help to expand the feasibility of the zygote-mediated generation of genome-modified animals.
-
zygote mediated generation of genome modified mice using streptococcus thermophilus 1 derived crispr cas system
Biochemical and Biophysical Research Communications, 2016Co-Authors: Wataru Fujii, Shigeru Kakuta, Shin Yoshioka, Shigeru Kyuwa, Koji Sugiura, Kunihiko NaitoAbstract:Mammalian zygote-mediated genome-engineering by CRISPR/Cas is currently used for the generation of genome-modified animals. Here we report that a Streptococcus thermophilus-1 derived orthologous CRISPR/Cas system, which recognizes the 5′-NNAGAA sequence as a Protospacer Adjacent Motif (PAM), is useful in mouse zygotes and is applicable for generating knockout mice (87.5%) and targeted knock-in mice (45.5%). The induced mutation could be inherited in the next generation. This novel CRISPR/Cas can expand the feasibility of the zygote-mediated generation of genome-modified animals that require an exact mutation design.
-
Zygote-mediated generation of genome-modified mice using Streptococcus thermophilus 1-derived CRISPR/Cas system.
Biochemical and biophysical research communications, 2016Co-Authors: Wataru Fujii, Shigeru Kakuta, Shin Yoshioka, Shigeru Kyuwa, Koji Sugiura, Kunihiko NaitoAbstract:Mammalian zygote-mediated genome-engineering by CRISPR/Cas is currently used for the generation of genome-modified animals. Here we report that a Streptococcus thermophilus-1 derived orthologous CRISPR/Cas system, which recognizes the 5'-NNAGAA sequence as a Protospacer Adjacent Motif (PAM), is useful in mouse zygotes and is applicable for generating knockout mice (87.5%) and targeted knock-in mice (45.5%). The induced mutation could be inherited in the next generation. This novel CRISPR/Cas can expand the feasibility of the zygote-mediated generation of genome-modified animals that require an exact mutation design.
Wataru Fujii - One of the best experts on this subject based on the ideXlab platform.
-
Generation of genetically modified mice using SpCas9-NG engineered nuclease
Scientific Reports, 2019Co-Authors: Wataru Fujii, Koji Sugiura, Arisa Ikeda, Haruka Ito, Takuya Kanke, Kunihiko NaitoAbstract:Although genetically modified mice can be generated with high efficiency by using CRISPR/Cas9-mediated genome editing in mouse zygotes, only the loci with a Protospacer-Adjacent Motif (PAM) sequence are targetable. The present study investigated the usability of engineered Streptococcus pyogenes Cas9 (SpCas9-NG) in mouse zygotes. In addition to the 5′-NGG sequence, SpCas9-NG recognized the 5′-NGA, 5′-NGC and 5′-NGT sequences in mouse zygotes as PAMs that were appropriate for the generation of knockout mice. Moreover, SpCas9-NG-mediated genome editing enabled the generation of knock-in mice untargetable by the conventional SpCas9 in mouse zygotes. These results suggest that SpCas9-NG-mediated genome editing in zygotes is available for the generation of knockout and knock-in mice at the locus corresponding to NGN-PAM.
-
Efficient Generation of Genome-Modified Mice Using Campylobacter jejuni-Derived CRISPR/Cas.
International journal of molecular sciences, 2017Co-Authors: Wataru Fujii, Koji Sugiura, Arisa Ikeda, Kunihiko NaitoAbstract:Mammalian zygote-mediated genome-engineering by Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas is currently used for the generation of genome-modified animals. Here, we report that a Campylobacter jejuni-derived orthologous CRISPR/Cas system recognizes a 5′-NNNVRYAC sequence as a Protospacer-Adjacent Motif in mouse zygotes, and is applicable for efficient generation of knockout mice. Moreover, this novel CRISPR/Cas can be used for zygote-mediated knock-in at a unique locus, suggesting that this system could help to expand the feasibility of the zygote-mediated generation of genome-modified animals.
-
zygote mediated generation of genome modified mice using streptococcus thermophilus 1 derived crispr cas system
Biochemical and Biophysical Research Communications, 2016Co-Authors: Wataru Fujii, Shigeru Kakuta, Shin Yoshioka, Shigeru Kyuwa, Koji Sugiura, Kunihiko NaitoAbstract:Mammalian zygote-mediated genome-engineering by CRISPR/Cas is currently used for the generation of genome-modified animals. Here we report that a Streptococcus thermophilus-1 derived orthologous CRISPR/Cas system, which recognizes the 5′-NNAGAA sequence as a Protospacer Adjacent Motif (PAM), is useful in mouse zygotes and is applicable for generating knockout mice (87.5%) and targeted knock-in mice (45.5%). The induced mutation could be inherited in the next generation. This novel CRISPR/Cas can expand the feasibility of the zygote-mediated generation of genome-modified animals that require an exact mutation design.
-
Zygote-mediated generation of genome-modified mice using Streptococcus thermophilus 1-derived CRISPR/Cas system.
Biochemical and biophysical research communications, 2016Co-Authors: Wataru Fujii, Shigeru Kakuta, Shin Yoshioka, Shigeru Kyuwa, Koji Sugiura, Kunihiko NaitoAbstract:Mammalian zygote-mediated genome-engineering by CRISPR/Cas is currently used for the generation of genome-modified animals. Here we report that a Streptococcus thermophilus-1 derived orthologous CRISPR/Cas system, which recognizes the 5'-NNAGAA sequence as a Protospacer Adjacent Motif (PAM), is useful in mouse zygotes and is applicable for generating knockout mice (87.5%) and targeted knock-in mice (45.5%). The induced mutation could be inherited in the next generation. This novel CRISPR/Cas can expand the feasibility of the zygote-mediated generation of genome-modified animals that require an exact mutation design.
Koji Sugiura - One of the best experts on this subject based on the ideXlab platform.
-
Generation of genetically modified mice using SpCas9-NG engineered nuclease
Scientific Reports, 2019Co-Authors: Wataru Fujii, Koji Sugiura, Arisa Ikeda, Haruka Ito, Takuya Kanke, Kunihiko NaitoAbstract:Although genetically modified mice can be generated with high efficiency by using CRISPR/Cas9-mediated genome editing in mouse zygotes, only the loci with a Protospacer-Adjacent Motif (PAM) sequence are targetable. The present study investigated the usability of engineered Streptococcus pyogenes Cas9 (SpCas9-NG) in mouse zygotes. In addition to the 5′-NGG sequence, SpCas9-NG recognized the 5′-NGA, 5′-NGC and 5′-NGT sequences in mouse zygotes as PAMs that were appropriate for the generation of knockout mice. Moreover, SpCas9-NG-mediated genome editing enabled the generation of knock-in mice untargetable by the conventional SpCas9 in mouse zygotes. These results suggest that SpCas9-NG-mediated genome editing in zygotes is available for the generation of knockout and knock-in mice at the locus corresponding to NGN-PAM.
-
Efficient Generation of Genome-Modified Mice Using Campylobacter jejuni-Derived CRISPR/Cas.
International journal of molecular sciences, 2017Co-Authors: Wataru Fujii, Koji Sugiura, Arisa Ikeda, Kunihiko NaitoAbstract:Mammalian zygote-mediated genome-engineering by Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas is currently used for the generation of genome-modified animals. Here, we report that a Campylobacter jejuni-derived orthologous CRISPR/Cas system recognizes a 5′-NNNVRYAC sequence as a Protospacer-Adjacent Motif in mouse zygotes, and is applicable for efficient generation of knockout mice. Moreover, this novel CRISPR/Cas can be used for zygote-mediated knock-in at a unique locus, suggesting that this system could help to expand the feasibility of the zygote-mediated generation of genome-modified animals.
-
zygote mediated generation of genome modified mice using streptococcus thermophilus 1 derived crispr cas system
Biochemical and Biophysical Research Communications, 2016Co-Authors: Wataru Fujii, Shigeru Kakuta, Shin Yoshioka, Shigeru Kyuwa, Koji Sugiura, Kunihiko NaitoAbstract:Mammalian zygote-mediated genome-engineering by CRISPR/Cas is currently used for the generation of genome-modified animals. Here we report that a Streptococcus thermophilus-1 derived orthologous CRISPR/Cas system, which recognizes the 5′-NNAGAA sequence as a Protospacer Adjacent Motif (PAM), is useful in mouse zygotes and is applicable for generating knockout mice (87.5%) and targeted knock-in mice (45.5%). The induced mutation could be inherited in the next generation. This novel CRISPR/Cas can expand the feasibility of the zygote-mediated generation of genome-modified animals that require an exact mutation design.
-
Zygote-mediated generation of genome-modified mice using Streptococcus thermophilus 1-derived CRISPR/Cas system.
Biochemical and biophysical research communications, 2016Co-Authors: Wataru Fujii, Shigeru Kakuta, Shin Yoshioka, Shigeru Kyuwa, Koji Sugiura, Kunihiko NaitoAbstract:Mammalian zygote-mediated genome-engineering by CRISPR/Cas is currently used for the generation of genome-modified animals. Here we report that a Streptococcus thermophilus-1 derived orthologous CRISPR/Cas system, which recognizes the 5'-NNAGAA sequence as a Protospacer Adjacent Motif (PAM), is useful in mouse zygotes and is applicable for generating knockout mice (87.5%) and targeted knock-in mice (45.5%). The induced mutation could be inherited in the next generation. This novel CRISPR/Cas can expand the feasibility of the zygote-mediated generation of genome-modified animals that require an exact mutation design.
Jennifer A Doudna - One of the best experts on this subject based on the ideXlab platform.
-
real time observation of dna recognition and rejection by the rna guided endonuclease cas9
Nature Communications, 2016Co-Authors: Digvijay Singh, Samuel H Sternberg, Jingyi Fei, Jennifer A DoudnaAbstract:Binding specificity of Cas9-guide RNA complexes to DNA is important for genome-engineering applications; however, how mismatches influence target recognition/rejection kinetics is not well understood. Here we used single-molecule FRET to probe real-time interactions between Cas9-RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence; however, the dissociation rate greatly increases from 2 s(-1) upon introduction of mismatches proximal to Protospacer-Adjacent Motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 11 base pairs in length, which prevent DNA cleavage, still allow formation of a stable complex (dissociation rate <0.006 s(-1)), suggesting that extremely slow rejection could sequester Cas9-RNA, increasing the Cas9 expression level necessary for genome-editing, thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading.
-
real time observation of dna recognition and rejection by the rna guided endonuclease cas9
bioRxiv, 2016Co-Authors: Digvijay Singh, Samuel H Sternberg, Jingyi Fei, Jennifer A DoudnaAbstract:Binding specificity of Cas9-guide RNA complexes to DNA is important for genome engineering applications, but how mismatches influence target recognition and rejection kinetics is not well understood. We used single-molecule FRET to probe real-time interactions between Cas9-RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence, but the dissociation rate greatly increases from 2 s-1 upon introduction of mismatches proximal to the Protospacer Adjacent Motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 12 base pairs in length, which prevent DNA cleavage, still allow the formation of a stable complex (off-rate
-
programmable rna recognition and cleavage by crispr cas9
Nature, 2014Co-Authors: Mitchell R Oconnell, Benjamin L Oakes, Samuel H Sternberg, Alexandra Eastseletsky, Matias Kaplan, Jennifer A DoudnaAbstract:In the presence of a short DNA oligonucleotide containing a Protospacer Adjacent Motif, a guide-RNA-programmed Cas9 is able to specifically bind and/or cleave single-stranded RNA—this system can be used to isolate specific endogenous RNA transcripts from a cell lysate without any tag or modification. The bacterial CRISPR immune defence system, and its effector Cas9 in particular, have recently been exploited for sequence-specific genome editing in eukaryotic cells. Cas9 binds a guide RNA and in the presence of a DNA Motif known as Protospacer Adjacent Motif (PAM), is able to cleave the target DNA. New work by Jennifer Doudna and colleagues reveals the unexpected result that in the presence of a DNA oligomer containing PAM, a guide RNA-programmed Cas9 is able to cleave single-stranded RNA as well. They show that this system can also be used to isolate specific endogenous RNA transcripts, without any tag or modification, from a cell lysate. Thus, the system can be programmed to either bind or cut desired RNA targets, depending on the PAM used. This work and points the way towards possible new technologies for programmable RNA recognition. The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA–DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage1,2,3,4,5. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the Protospacer Adjacent Motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA4,5,6,7. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms8, but it has been thought to be incapable of targeting RNA5. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage7. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.
-
programmable rna recognition and cleavage by crispr cas9
Nature, 2014Co-Authors: Mitchell R Oconnell, Benjamin L Oakes, Samuel H Sternberg, Alexandra Eastseletsky, Matias Kaplan, Jennifer A DoudnaAbstract:In the presence of a short DNA oligonucleotide containing a Protospacer Adjacent Motif, a guide-RNA-programmed Cas9 is able to specifically bind and/or cleave single-stranded RNA—this system can be used to isolate specific endogenous RNA transcripts from a cell lysate without any tag or modification.
Yannick Doyon - One of the best experts on this subject based on the ideXlab platform.
-
Rewired Cas9s with Minimal Sequence Constraints.
Trends in pharmacological sciences, 2020Co-Authors: Sébastien Lévesque, Daniel Agudelo, Yannick DoyonAbstract:The genome editing toolkit is ever expanding. Although CRISPR-Cas systems can target virtually any gene, single-nucleotide resolution is yet to be achieved. Walton and colleagues engineered nucleases and base editors compatible with every Protospacer Adjacent Motif (PAM) to achieve high-precision targeting. Their findings revealed the striking plasticity of Cas9.
-
versatile in vivo genome editing with the prototypical streptococcus thermophilus crispr1 cas9 system
bioRxiv, 2018Co-Authors: Sophie Carter, Daniel Agudelo, Alexis Duringer, Jeremy Loehr, Yannick DoyonAbstract:Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique Protospacer Adjacent Motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.
