Limits...
Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos.

Wang L, Shao Y, Guan Y, Li L, Wu L, Chen F, Liu M, Chen H, Ma Y, Ma X, Liu M, Li D - Sci Rep (2015)

Bottom Line: The CRISPR-Cas RNA-guided system has versatile uses in many organisms and allows modification of multiple target sites simultaneously.Through the injection of Cas9 protein instead of mRNA into embryos, we observed fewer off-target effects of Cas9 and increased point mutation knock-in efficiency.In addition, we combined the Cre-Loxp system with a gene-trap strategy to insert a GFP reporter in the reverse orientation into the rat Lgr5 locus, which was later inverted by Cre-mediated recombination, yielding a conditional knockout/reporter strategy suitable for mosaic mutation analysis.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.

ABSTRACT
The CRISPR-Cas RNA-guided system has versatile uses in many organisms and allows modification of multiple target sites simultaneously. Generating novel genetically modified mouse and rat models is one valuable application of this system. Through the injection of Cas9 protein instead of mRNA into embryos, we observed fewer off-target effects of Cas9 and increased point mutation knock-in efficiency. Large genomic DNA fragment (up to 95 kb) deletion mice were generated for in vivo study of lncRNAs and gene clusters. Site-specific insertion of a 2.7 kb CreERT2 cassette into the mouse Nfatc1 locus allowed labeling and tracing of hair follicle stem cells. In addition, we combined the Cre-Loxp system with a gene-trap strategy to insert a GFP reporter in the reverse orientation into the rat Lgr5 locus, which was later inverted by Cre-mediated recombination, yielding a conditional knockout/reporter strategy suitable for mosaic mutation analysis.

No MeSH data available.


Related in: MedlinePlus

Off-target analysis of sgRNA targeting the AR genomic locus.(a) Genomic DNA extracted from 12 F0 pups was subjected to PCR with on-target primers and subsequent T7E1 digestion. On-target mutants are indicated by arrowheads. M, DNA marker. (b) T7E1 digestion analysis of founders on two off-target (OT) sites. The sequence of On-target and OT sites are listed. The PAM sequences are in red and mismatched nucleotides are in blue. (c) The sequences of the OT sites of the founders bearing mutations are listed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4664917&req=5

f2: Off-target analysis of sgRNA targeting the AR genomic locus.(a) Genomic DNA extracted from 12 F0 pups was subjected to PCR with on-target primers and subsequent T7E1 digestion. On-target mutants are indicated by arrowheads. M, DNA marker. (b) T7E1 digestion analysis of founders on two off-target (OT) sites. The sequence of On-target and OT sites are listed. The PAM sequences are in red and mismatched nucleotides are in blue. (c) The sequences of the OT sites of the founders bearing mutations are listed.

Mentions: It was reported that reducing the amount of Cas9/sgRNA plasmid DNA during transfection increased the cleavage specificity30. As Cas9 protein exhibits a shorter duration than DNA/RNA transfected into the cell, we assumed that injection of Cas9 protein could be helpful to increase the targeting specificity. To check for potential off target activities by Cas9 protein, we chose a previously reported sgRNA that targets the mouse androgen receptor (Ar) locus31. After injection of Cas9 protein:sgRNA, 3 on-target mutant F0 pups were identified by T7E1 digestion (Fig. 2a). The efficiency was comparable with a previous report31. Two potential off-target sites were analyzed by T7E1 assay followed by sequencing (Fig. 2b,c). For each off-target site, 1 of 3 founders exhibited off-target digestion (Fig. 2b,c). It was reported that all 3 on-target founders contained both of these two off-target mutations in mice generated via injection of Cas9 mRNA31. The off-target frequency was reduced when we injected Cas9 protein, but the on target efficiency was consistent with previous reports17. This is probably due to the shorter half-life of Cas9 protein compared to Cas9 mRNA and its subsequent translation1732. The results suggested that Cas9 protein injection could be used as an alternative way to generate mutations with the potential benefit of reducing off-target digestion.


Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos.

Wang L, Shao Y, Guan Y, Li L, Wu L, Chen F, Liu M, Chen H, Ma Y, Ma X, Liu M, Li D - Sci Rep (2015)

Off-target analysis of sgRNA targeting the AR genomic locus.(a) Genomic DNA extracted from 12 F0 pups was subjected to PCR with on-target primers and subsequent T7E1 digestion. On-target mutants are indicated by arrowheads. M, DNA marker. (b) T7E1 digestion analysis of founders on two off-target (OT) sites. The sequence of On-target and OT sites are listed. The PAM sequences are in red and mismatched nucleotides are in blue. (c) The sequences of the OT sites of the founders bearing mutations are listed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664917&req=5

f2: Off-target analysis of sgRNA targeting the AR genomic locus.(a) Genomic DNA extracted from 12 F0 pups was subjected to PCR with on-target primers and subsequent T7E1 digestion. On-target mutants are indicated by arrowheads. M, DNA marker. (b) T7E1 digestion analysis of founders on two off-target (OT) sites. The sequence of On-target and OT sites are listed. The PAM sequences are in red and mismatched nucleotides are in blue. (c) The sequences of the OT sites of the founders bearing mutations are listed.
Mentions: It was reported that reducing the amount of Cas9/sgRNA plasmid DNA during transfection increased the cleavage specificity30. As Cas9 protein exhibits a shorter duration than DNA/RNA transfected into the cell, we assumed that injection of Cas9 protein could be helpful to increase the targeting specificity. To check for potential off target activities by Cas9 protein, we chose a previously reported sgRNA that targets the mouse androgen receptor (Ar) locus31. After injection of Cas9 protein:sgRNA, 3 on-target mutant F0 pups were identified by T7E1 digestion (Fig. 2a). The efficiency was comparable with a previous report31. Two potential off-target sites were analyzed by T7E1 assay followed by sequencing (Fig. 2b,c). For each off-target site, 1 of 3 founders exhibited off-target digestion (Fig. 2b,c). It was reported that all 3 on-target founders contained both of these two off-target mutations in mice generated via injection of Cas9 mRNA31. The off-target frequency was reduced when we injected Cas9 protein, but the on target efficiency was consistent with previous reports17. This is probably due to the shorter half-life of Cas9 protein compared to Cas9 mRNA and its subsequent translation1732. The results suggested that Cas9 protein injection could be used as an alternative way to generate mutations with the potential benefit of reducing off-target digestion.

Bottom Line: The CRISPR-Cas RNA-guided system has versatile uses in many organisms and allows modification of multiple target sites simultaneously.Through the injection of Cas9 protein instead of mRNA into embryos, we observed fewer off-target effects of Cas9 and increased point mutation knock-in efficiency.In addition, we combined the Cre-Loxp system with a gene-trap strategy to insert a GFP reporter in the reverse orientation into the rat Lgr5 locus, which was later inverted by Cre-mediated recombination, yielding a conditional knockout/reporter strategy suitable for mosaic mutation analysis.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.

ABSTRACT
The CRISPR-Cas RNA-guided system has versatile uses in many organisms and allows modification of multiple target sites simultaneously. Generating novel genetically modified mouse and rat models is one valuable application of this system. Through the injection of Cas9 protein instead of mRNA into embryos, we observed fewer off-target effects of Cas9 and increased point mutation knock-in efficiency. Large genomic DNA fragment (up to 95 kb) deletion mice were generated for in vivo study of lncRNAs and gene clusters. Site-specific insertion of a 2.7 kb CreERT2 cassette into the mouse Nfatc1 locus allowed labeling and tracing of hair follicle stem cells. In addition, we combined the Cre-Loxp system with a gene-trap strategy to insert a GFP reporter in the reverse orientation into the rat Lgr5 locus, which was later inverted by Cre-mediated recombination, yielding a conditional knockout/reporter strategy suitable for mosaic mutation analysis.

No MeSH data available.


Related in: MedlinePlus