spcas9 sgrna Search Results


px330 flag wt spcas9  (Addgene inc)
93
Addgene inc px330 flag wt spcas9
Px330 Flag Wt Spcas9, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pbluescript sgrna expression plasmids  (Addgene inc)
93
Addgene inc pbluescript sgrna expression plasmids
a) <t>Nme1Cas9/sgRNA/DNA</t> ternary complex structure, PDB:6JDV. <t>Nme2Cas9</t> is 98% identical to Nme2Cas9 outside of the WED and PAM-interacting domains. Black spheres represent N- and C-termini and colored spheres represent sites of domain insertion. Deaminase domain insertion sites (Nme2Cas9 aa numbers) are specified to the right, with colors matching the sites indicated in the structure. b) Activities of Nme2-ABE8e constructs in mCherry reporter cells (activated upon A-to-G editing) after plasmid transfection, measured by flow cytometry (n = 3 biological replicates in technical duplicate; data represent mean ± SD). c) A-to-G editing following transfection of Spy-ABE8e vs. Nme2-ABE8e plasmids, using PAM-matched, endogenous HEK293T genomic loci. The editing efficiency at the maximally edited adenine for each target was plotted. Editing efficiencies were measured by amplicon deep sequencing (n = 3 biological replicates; data represent mean ± SD). d) Data from (c) were aggregated and replotted, with each data point representing the maximum A-to-G editing efficiency of an individual target site, as measured by amplicon deep sequencing (n = 3 biological replicates; data represent mean ± SEM). e) Summary of mean A-to-G editing activities and editing windows for Spy- and Nme2-ABE8e constructs in HEK293T cells. Numbers provided for each position in the protospacer represent the mean A-to-G editing efficiency across eight PAM-matched endogenous target sites, as measured via amplicon deep sequencing (n = 3 biological replicates). Crossed-out boxes indicate that no adenine was present at the specified position in the target panel tested.
Pbluescript Sgrna Expression Plasmids, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/spcas9+sgrna/SpCas9_sgRNA_expression_in_pBluescript+(Plasmid+%23122089)/pmc10153126-167-11-15
Average 93 stars, based on 1 article reviews
pbluescript sgrna expression plasmids - by Bioz Stars, 2026-09
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px330 flag sniper spcas9  (Addgene inc)
91
Addgene inc px330 flag sniper spcas9
a X-ray crystallography derived structure of <t>SpCas9-sgRNA-DNA</t> complex in the conformation closest to the cleavage-competent state (PDB ID: 5f9r) . b Sequences of SpCas9-HF1 and the selected Blackjack-SpCas9-HF1 at the region affected, between residues L1004 and D1017; deletions (−) and insertions (green) are indicated. See also Supplementary Fig. . c , d Blackjack mutations increase on-target activities of increased fidelity variants with 21G-sgRNAs on different targets. Means are shown, error bars represent the standard deviation (s.d.) for n = 3 biologically independent samples (overlaid as white circles).
Px330 Flag Sniper Spcas9, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/spcas9+sgrna/pX330-Flag-Sniper+SpCas9+(without+sgRNA%3B+with+silent+mutations)+(Plasmid+%23126777)/pmc07060260-191-61-68
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px330 flag spcas9 hf1  (Addgene inc)
86
Addgene inc px330 flag spcas9 hf1
a X-ray crystallography derived structure of <t>SpCas9-sgRNA-DNA</t> complex in the conformation closest to the cleavage-competent state (PDB ID: 5f9r) . b Sequences of SpCas9-HF1 and the selected Blackjack-SpCas9-HF1 at the region affected, between residues L1004 and D1017; deletions (−) and insertions (green) are indicated. See also Supplementary Fig. . c , d Blackjack mutations increase on-target activities of increased fidelity variants with 21G-sgRNAs on different targets. Means are shown, error bars represent the standard deviation (s.d.) for n = 3 biologically independent samples (overlaid as white circles).
Px330 Flag Spcas9 Hf1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/spcas9+sgrna/pX330-Flag-SpCas9-HF1+(without+sgRNA)+(Plasmid+%2392102)/pmc06389135-209-23-26
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px330 flag spcas9 hf1 - by Bioz Stars, 2026-09
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msp3485  (Addgene inc)
93
Addgene inc msp3485
a X-ray crystallography derived structure of <t>SpCas9-sgRNA-DNA</t> complex in the conformation closest to the cleavage-competent state (PDB ID: 5f9r) . b Sequences of SpCas9-HF1 and the selected Blackjack-SpCas9-HF1 at the region affected, between residues L1004 and D1017; deletions (−) and insertions (green) are indicated. See also Supplementary Fig. . c , d Blackjack mutations increase on-target activities of increased fidelity variants with 21G-sgRNAs on different targets. Means are shown, error bars represent the standard deviation (s.d.) for n = 3 biologically independent samples (overlaid as white circles).
Msp3485, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/spcas9+sgrna/pT7-%5BBsaI_cassette%5D-SpCas9_sgRNA_scaffold+(MSP3485)+(Plasmid+%23140082)/pmc09043867-220-28-30
Average 93 stars, based on 1 article reviews
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px330 flag hifi spcas9  (Addgene inc)
93
Addgene inc px330 flag hifi spcas9
a X-ray crystallography derived structure of <t>SpCas9-sgRNA-DNA</t> complex in the conformation closest to the cleavage-competent state (PDB ID: 5f9r) . b Sequences of SpCas9-HF1 and the selected Blackjack-SpCas9-HF1 at the region affected, between residues L1004 and D1017; deletions (−) and insertions (green) are indicated. See also Supplementary Fig. . c , d Blackjack mutations increase on-target activities of increased fidelity variants with 21G-sgRNAs on different targets. Means are shown, error bars represent the standard deviation (s.d.) for n = 3 biologically independent samples (overlaid as white circles).
Px330 Flag Hifi Spcas9, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/spcas9+sgrna/pX330-Flag-HiFi+SpCas9+(without+sgRNA%3B+with+silent+mutations)+(Plasmid+%23126778)/pmc08934663-60-12-19
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px330 flag hifi spcas9 - by Bioz Stars, 2026-09
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px552 cmv mcherry u6 spcas9 sgrna scaffold  (Addgene inc)
91
Addgene inc px552 cmv mcherry u6 spcas9 sgrna scaffold
a X-ray crystallography derived structure of <t>SpCas9-sgRNA-DNA</t> complex in the conformation closest to the cleavage-competent state (PDB ID: 5f9r) . b Sequences of SpCas9-HF1 and the selected Blackjack-SpCas9-HF1 at the region affected, between residues L1004 and D1017; deletions (−) and insertions (green) are indicated. See also Supplementary Fig. . c , d Blackjack mutations increase on-target activities of increased fidelity variants with 21G-sgRNAs on different targets. Means are shown, error bars represent the standard deviation (s.d.) for n = 3 biologically independent samples (overlaid as white circles).
Px552 Cmv Mcherry U6 Spcas9 Sgrna Scaffold, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/spcas9+sgrna/pX552-CMV-mCherry-U6-(SpCas9)sgRNA+scaffold+(Plasmid+%23107051)/pmc07519688-130-14-16
Average 91 stars, based on 1 article reviews
px552 cmv mcherry u6 spcas9 sgrna scaffold - by Bioz Stars, 2026-09
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spcas9 sgrna platform  (Addgene inc)
91
Addgene inc spcas9 sgrna platform
a X-ray crystallography derived structure of <t>SpCas9-sgRNA-DNA</t> complex in the conformation closest to the cleavage-competent state (PDB ID: 5f9r) . b Sequences of SpCas9-HF1 and the selected Blackjack-SpCas9-HF1 at the region affected, between residues L1004 and D1017; deletions (−) and insertions (green) are indicated. See also Supplementary Fig. . c , d Blackjack mutations increase on-target activities of increased fidelity variants with 21G-sgRNAs on different targets. Means are shown, error bars represent the standard deviation (s.d.) for n = 3 biologically independent samples (overlaid as white circles).
Spcas9 Sgrna Platform, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/spcas9+sgrna/pX552-SpCas9+sgRNA-LacZ+sgRNA+(Plasmid+%23107028)/bio_rxiv__2024__05__13__593900-166-11-23
Average 91 stars, based on 1 article reviews
spcas9 sgrna platform - by Bioz Stars, 2026-09
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lipid nanoparticle containing sgrna and spcas9 mrna  (Regeneron inc)
90
Regeneron inc lipid nanoparticle containing sgrna and spcas9 mrna
A) Overall Structure of the <t>Cas9-sgRNA-DNA</t> Ternary Complex. Ribbon representation of the Cas9-sgRNA-DNA complex. Disordered linkers are shown as red dotted lines. Reproduced with permission from PMID: 24529477. B) 3-D Structure of <t>the</t> <t>SpCas9</t> protein, showing the location of the identified immunodominant epitoes α and β. Reproduced with permission from PMID: 31015529.
Lipid Nanoparticle Containing Sgrna And Spcas9 Mrna, supplied by Regeneron inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/spcas9+sgrna/lipid+nanoparticle+containing+sgrna+and+spcas9+mrna/pmc07115921-5-24-35
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lipid nanoparticle containing sgrna and spcas9 mrna - by Bioz Stars, 2026-09
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spcas9-sgrna plasmids  (PolyScience)
90
PolyScience spcas9-sgrna plasmids
A) Overall Structure of the <t>Cas9-sgRNA-DNA</t> Ternary Complex. Ribbon representation of the Cas9-sgRNA-DNA complex. Disordered linkers are shown as red dotted lines. Reproduced with permission from PMID: 24529477. B) 3-D Structure of <t>the</t> <t>SpCas9</t> protein, showing the location of the identified immunodominant epitoes α and β. Reproduced with permission from PMID: 31015529.
Spcas9 Sgrna Plasmids, supplied by PolyScience, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/spcas9+sgrna/spcas9+sgrna+plasmids/pmc10657421-423-1-8
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spcas9-sgrna plasmids - by Bioz Stars, 2026-09
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px552-spcas9 sgrna-yfp sgrna2 (plasmid #107029)  (Addgene inc)
N/A
Standard format: Plasmid sent in bacteria as agar stab
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Image Search Results


a) Nme1Cas9/sgRNA/DNA ternary complex structure, PDB:6JDV. Nme2Cas9 is 98% identical to Nme2Cas9 outside of the WED and PAM-interacting domains. Black spheres represent N- and C-termini and colored spheres represent sites of domain insertion. Deaminase domain insertion sites (Nme2Cas9 aa numbers) are specified to the right, with colors matching the sites indicated in the structure. b) Activities of Nme2-ABE8e constructs in mCherry reporter cells (activated upon A-to-G editing) after plasmid transfection, measured by flow cytometry (n = 3 biological replicates in technical duplicate; data represent mean ± SD). c) A-to-G editing following transfection of Spy-ABE8e vs. Nme2-ABE8e plasmids, using PAM-matched, endogenous HEK293T genomic loci. The editing efficiency at the maximally edited adenine for each target was plotted. Editing efficiencies were measured by amplicon deep sequencing (n = 3 biological replicates; data represent mean ± SD). d) Data from (c) were aggregated and replotted, with each data point representing the maximum A-to-G editing efficiency of an individual target site, as measured by amplicon deep sequencing (n = 3 biological replicates; data represent mean ± SEM). e) Summary of mean A-to-G editing activities and editing windows for Spy- and Nme2-ABE8e constructs in HEK293T cells. Numbers provided for each position in the protospacer represent the mean A-to-G editing efficiency across eight PAM-matched endogenous target sites, as measured via amplicon deep sequencing (n = 3 biological replicates). Crossed-out boxes indicate that no adenine was present at the specified position in the target panel tested.

Journal: bioRxiv

Article Title: Engineering Nme2Cas9 Adenine Base Editors with Improved Activity and Targeting Scope

doi: 10.1101/2023.04.14.536905

Figure Lengend Snippet: a) Nme1Cas9/sgRNA/DNA ternary complex structure, PDB:6JDV. Nme2Cas9 is 98% identical to Nme2Cas9 outside of the WED and PAM-interacting domains. Black spheres represent N- and C-termini and colored spheres represent sites of domain insertion. Deaminase domain insertion sites (Nme2Cas9 aa numbers) are specified to the right, with colors matching the sites indicated in the structure. b) Activities of Nme2-ABE8e constructs in mCherry reporter cells (activated upon A-to-G editing) after plasmid transfection, measured by flow cytometry (n = 3 biological replicates in technical duplicate; data represent mean ± SD). c) A-to-G editing following transfection of Spy-ABE8e vs. Nme2-ABE8e plasmids, using PAM-matched, endogenous HEK293T genomic loci. The editing efficiency at the maximally edited adenine for each target was plotted. Editing efficiencies were measured by amplicon deep sequencing (n = 3 biological replicates; data represent mean ± SD). d) Data from (c) were aggregated and replotted, with each data point representing the maximum A-to-G editing efficiency of an individual target site, as measured by amplicon deep sequencing (n = 3 biological replicates; data represent mean ± SEM). e) Summary of mean A-to-G editing activities and editing windows for Spy- and Nme2-ABE8e constructs in HEK293T cells. Numbers provided for each position in the protospacer represent the mean A-to-G editing efficiency across eight PAM-matched endogenous target sites, as measured via amplicon deep sequencing (n = 3 biological replicates). Crossed-out boxes indicate that no adenine was present at the specified position in the target panel tested.

Article Snippet: U6-driven sgRNA plasmids for the various Cas effectors were cloned using pBluescript sgRNA expression plasmids (Addgene #122089, #122090, #122091 for SpyCas9, SauCas9 and Nme2Cas9 respectively).

Techniques: Construct, Plasmid Preparation, Transfection, Flow Cytometry, Amplification, Sequencing

a X-ray crystallography derived structure of SpCas9-sgRNA-DNA complex in the conformation closest to the cleavage-competent state (PDB ID: 5f9r) . b Sequences of SpCas9-HF1 and the selected Blackjack-SpCas9-HF1 at the region affected, between residues L1004 and D1017; deletions (−) and insertions (green) are indicated. See also Supplementary Fig. . c , d Blackjack mutations increase on-target activities of increased fidelity variants with 21G-sgRNAs on different targets. Means are shown, error bars represent the standard deviation (s.d.) for n = 3 biologically independent samples (overlaid as white circles).

Journal: Nature Communications

Article Title: Blackjack mutations improve the on-target activities of increased fidelity variants of SpCas9 with 5′G-extended sgRNAs

doi: 10.1038/s41467-020-15021-5

Figure Lengend Snippet: a X-ray crystallography derived structure of SpCas9-sgRNA-DNA complex in the conformation closest to the cleavage-competent state (PDB ID: 5f9r) . b Sequences of SpCas9-HF1 and the selected Blackjack-SpCas9-HF1 at the region affected, between residues L1004 and D1017; deletions (−) and insertions (green) are indicated. See also Supplementary Fig. . c , d Blackjack mutations increase on-target activities of increased fidelity variants with 21G-sgRNAs on different targets. Means are shown, error bars represent the standard deviation (s.d.) for n = 3 biologically independent samples (overlaid as white circles).

Article Snippet: Plasmids developed by us and deposited at Addgene are the following: pX330-Flag-dSpCas9 (Addgene #92113), pX330-Flag-WT_SpCas9 (without sgRNA; with silent mutations) (Addgene #126753), pX330-Flag-eSpCas9 (without sgRNA; with silent mutations) (Addgene #126754), pX330-Flag-SpCas9-HF1 (without sgRNA; with silent mutations) (Addgene #126755), pX330-Flag-HypaSpCas9 (without sgRNA; with silent mutations) (Addgene #126756),pX330-Flag-evoSpCas9 (without sgRNA; with silent mutations) (Addgene #126758), pX330-Flag-HeFSpCas9 (without sgRNA; with silent mutations) (Addgene #126759), pX330-Flag-Sniper SpCas9 (without sgRNA; with silent mutations) (Addgene #126777), pX330-Flag-HiFi SpCas9 (without sgRNA; with silent mutations) (Addgene #126778), B-SpCas9 (Addgene #126760), B-eSpCas9 (Addgene #126761), B-SpCas9-HF1 (Addgene #126762), B-HypaSpCas9 (Addgene #126763), B-evoSpCas9 (Addgene #126765), B-HeFSpCas9 (Addgene #126766) eSpCas9-plus (Addgene #126767), SpCas9-HF1-plus (Addgene #126768) pET-FLAG-eSpCas9 (Addgene #126769), pET-FLAG-SpCas9-HF1 (Addgene #126770), pET-FLAG-B-eSpCas9 (Addgene #126772), pET-FLAG-eSpCas9-plus (Addgene #126774), pET-FLAG-SpCas9-HF1-plus (Addgene #126775) pmCherry_sgRNA-ver2 (Addgene #126776), pmCherry_gRNA (Addgene: #80457)

Techniques: Derivative Assay, Standard Deviation

a Blackjack mutations increase the target-selectivity of their respective parent SpCas9 variants. EGFP-disruption activities with perfectly matching 20G-sgRNAs. Results are shown only for those target sites where the SpCas9 variant without Blackjack mutations exhibits higher than background level cleavage. See also Supplementary Fig. . b On-target activities with 21G-sgRNAs on more target sites for which the SpCas9 variant with Blackjack mutations using 20G-sgRNAs exhibits at least 70% on-target activity compared to WT SpCas9. No target corresponds to this condition in the case of HeFSpCas9. See also Supplementary Fig. . a , b The median and the interquartile range are shown; data points are plotted as open circles representing the mean of biologically independent triplicates. Spacers are schematically depicted beside the charts as combs: green color teeth indicate matching-, while a red color tooth indicates the presence of an appended nucleotide within the spacer; numbering of tooth position corresponds to the distance of the nucleotide from the PAM; the starting 20th nucleotide of the spacer is indicated by an uppercase letter and an appended 21st nucleotide by a red lowercase letter. Statistical significance was assessed using two-sided Paired-samples Student’s t -test or two-sided Wilcoxon signed ranks test as appropriate; ns not significant. A summary of data distributions and statistical details is reported in Supplementary Data .

Journal: Nature Communications

Article Title: Blackjack mutations improve the on-target activities of increased fidelity variants of SpCas9 with 5′G-extended sgRNAs

doi: 10.1038/s41467-020-15021-5

Figure Lengend Snippet: a Blackjack mutations increase the target-selectivity of their respective parent SpCas9 variants. EGFP-disruption activities with perfectly matching 20G-sgRNAs. Results are shown only for those target sites where the SpCas9 variant without Blackjack mutations exhibits higher than background level cleavage. See also Supplementary Fig. . b On-target activities with 21G-sgRNAs on more target sites for which the SpCas9 variant with Blackjack mutations using 20G-sgRNAs exhibits at least 70% on-target activity compared to WT SpCas9. No target corresponds to this condition in the case of HeFSpCas9. See also Supplementary Fig. . a , b The median and the interquartile range are shown; data points are plotted as open circles representing the mean of biologically independent triplicates. Spacers are schematically depicted beside the charts as combs: green color teeth indicate matching-, while a red color tooth indicates the presence of an appended nucleotide within the spacer; numbering of tooth position corresponds to the distance of the nucleotide from the PAM; the starting 20th nucleotide of the spacer is indicated by an uppercase letter and an appended 21st nucleotide by a red lowercase letter. Statistical significance was assessed using two-sided Paired-samples Student’s t -test or two-sided Wilcoxon signed ranks test as appropriate; ns not significant. A summary of data distributions and statistical details is reported in Supplementary Data .

Article Snippet: Plasmids developed by us and deposited at Addgene are the following: pX330-Flag-dSpCas9 (Addgene #92113), pX330-Flag-WT_SpCas9 (without sgRNA; with silent mutations) (Addgene #126753), pX330-Flag-eSpCas9 (without sgRNA; with silent mutations) (Addgene #126754), pX330-Flag-SpCas9-HF1 (without sgRNA; with silent mutations) (Addgene #126755), pX330-Flag-HypaSpCas9 (without sgRNA; with silent mutations) (Addgene #126756),pX330-Flag-evoSpCas9 (without sgRNA; with silent mutations) (Addgene #126758), pX330-Flag-HeFSpCas9 (without sgRNA; with silent mutations) (Addgene #126759), pX330-Flag-Sniper SpCas9 (without sgRNA; with silent mutations) (Addgene #126777), pX330-Flag-HiFi SpCas9 (without sgRNA; with silent mutations) (Addgene #126778), B-SpCas9 (Addgene #126760), B-eSpCas9 (Addgene #126761), B-SpCas9-HF1 (Addgene #126762), B-HypaSpCas9 (Addgene #126763), B-evoSpCas9 (Addgene #126765), B-HeFSpCas9 (Addgene #126766) eSpCas9-plus (Addgene #126767), SpCas9-HF1-plus (Addgene #126768) pET-FLAG-eSpCas9 (Addgene #126769), pET-FLAG-SpCas9-HF1 (Addgene #126770), pET-FLAG-B-eSpCas9 (Addgene #126772), pET-FLAG-eSpCas9-plus (Addgene #126774), pET-FLAG-SpCas9-HF1-plus (Addgene #126775) pmCherry_sgRNA-ver2 (Addgene #126776), pmCherry_gRNA (Addgene: #80457)

Techniques: Disruption, Variant Assay, Activity Assay

a Blackjack mutations increase the fidelity of their respective parent SpCas9 variants. EGFP-disruption activities with partially mismatching 20G-sgRNAs. Results are shown only for those target sites where both the non-Blackjack parent- and Blackjack-SpCas9 variant exhibit at least 70% on-target activity (with perfectly matching 20G-sgRNAs) compared to WT SpCas9. Only one target (with three mismatched positions) matches this condition in the case of evo- or HeFSpCas9. The median and the interquartile range are shown; data points are plotted as open circles representing the mean of biologically independent triplicates. Spacers are schematically depicted beside the charts as combs: green color teeth indicate matching-, while a red color tooth indicates the presence of a mismatching nucleotide (not necessarily the exact position) within the spacer; numbering of the tooth positions corresponds to the distance of the nucleotide from the PAM; the starting 20th nucleotide of the spacer is indicated by an uppercase letter. Statistical significance was assessed using two-sided Paired-samples Student’s t -test or two-sided Wilcoxon signed ranks test as appropriate; ns not significant. Summary of data distributions and statistical details are reported in Supplementary Data . See also Supplementary Fig. . b Bar chart of the total number of off-target sites detected by GUIDE-seq for WT and B-SpCas9 variants on six target sites targeted with 20G- or 21G-sgRNAs. See also Supplementary Fig. .

Journal: Nature Communications

Article Title: Blackjack mutations improve the on-target activities of increased fidelity variants of SpCas9 with 5′G-extended sgRNAs

doi: 10.1038/s41467-020-15021-5

Figure Lengend Snippet: a Blackjack mutations increase the fidelity of their respective parent SpCas9 variants. EGFP-disruption activities with partially mismatching 20G-sgRNAs. Results are shown only for those target sites where both the non-Blackjack parent- and Blackjack-SpCas9 variant exhibit at least 70% on-target activity (with perfectly matching 20G-sgRNAs) compared to WT SpCas9. Only one target (with three mismatched positions) matches this condition in the case of evo- or HeFSpCas9. The median and the interquartile range are shown; data points are plotted as open circles representing the mean of biologically independent triplicates. Spacers are schematically depicted beside the charts as combs: green color teeth indicate matching-, while a red color tooth indicates the presence of a mismatching nucleotide (not necessarily the exact position) within the spacer; numbering of the tooth positions corresponds to the distance of the nucleotide from the PAM; the starting 20th nucleotide of the spacer is indicated by an uppercase letter. Statistical significance was assessed using two-sided Paired-samples Student’s t -test or two-sided Wilcoxon signed ranks test as appropriate; ns not significant. Summary of data distributions and statistical details are reported in Supplementary Data . See also Supplementary Fig. . b Bar chart of the total number of off-target sites detected by GUIDE-seq for WT and B-SpCas9 variants on six target sites targeted with 20G- or 21G-sgRNAs. See also Supplementary Fig. .

Article Snippet: Plasmids developed by us and deposited at Addgene are the following: pX330-Flag-dSpCas9 (Addgene #92113), pX330-Flag-WT_SpCas9 (without sgRNA; with silent mutations) (Addgene #126753), pX330-Flag-eSpCas9 (without sgRNA; with silent mutations) (Addgene #126754), pX330-Flag-SpCas9-HF1 (without sgRNA; with silent mutations) (Addgene #126755), pX330-Flag-HypaSpCas9 (without sgRNA; with silent mutations) (Addgene #126756),pX330-Flag-evoSpCas9 (without sgRNA; with silent mutations) (Addgene #126758), pX330-Flag-HeFSpCas9 (without sgRNA; with silent mutations) (Addgene #126759), pX330-Flag-Sniper SpCas9 (without sgRNA; with silent mutations) (Addgene #126777), pX330-Flag-HiFi SpCas9 (without sgRNA; with silent mutations) (Addgene #126778), B-SpCas9 (Addgene #126760), B-eSpCas9 (Addgene #126761), B-SpCas9-HF1 (Addgene #126762), B-HypaSpCas9 (Addgene #126763), B-evoSpCas9 (Addgene #126765), B-HeFSpCas9 (Addgene #126766) eSpCas9-plus (Addgene #126767), SpCas9-HF1-plus (Addgene #126768) pET-FLAG-eSpCas9 (Addgene #126769), pET-FLAG-SpCas9-HF1 (Addgene #126770), pET-FLAG-B-eSpCas9 (Addgene #126772), pET-FLAG-eSpCas9-plus (Addgene #126774), pET-FLAG-SpCas9-HF1-plus (Addgene #126775) pmCherry_sgRNA-ver2 (Addgene #126776), pmCherry_gRNA (Addgene: #80457)

Techniques: Disruption, Variant Assay, Activity Assay

a – c EGFP-disruption activity a with 20G-sgRNAs targeting 25 sites; b , c with either 20G- or 21G-sgRNA pairs targeting two alternative sets of 10 different sequences shown as the ratio of variant activity to WT activity. d , e On-target activities of SpCas9 variants across 23 endogenous target sites within the human VEGFA or FANCF loci targeted with d 20G- or e 21G-sgRNAs, measured by amplicon resequencing. f Bar chart of the total number of off-target sites detected by GUIDE-seq for SpCas9 variants on seven sites targeted with 20G-sgRNAs. a – e Tukey-type boxplots by BoxPlotR : center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the “minimum” and “maximum” data situated within 1.5 times the interquartile range from the 25th and 75th percentiles, respectively; notches indicate the 95% confidence intervals for the medians; crosses represent sample means; data points are plotted as open circles representing the mean of biologically independent triplicates. Spacers are schematically depicted beside the charts as combs: green color teeth indicate matching-, while a red color tooth indicates the presence of an appended nucleotide within the spacer; numbering of tooth position corresponds to the distance of the nucleotide from the PAM; the starting 20th nucleotide of the spacer is indicated by an uppercase letter and an appended 21st nucleotide by a red lowercase letter. See also Supplementary Figs. and .

Journal: Nature Communications

Article Title: Blackjack mutations improve the on-target activities of increased fidelity variants of SpCas9 with 5′G-extended sgRNAs

doi: 10.1038/s41467-020-15021-5

Figure Lengend Snippet: a – c EGFP-disruption activity a with 20G-sgRNAs targeting 25 sites; b , c with either 20G- or 21G-sgRNA pairs targeting two alternative sets of 10 different sequences shown as the ratio of variant activity to WT activity. d , e On-target activities of SpCas9 variants across 23 endogenous target sites within the human VEGFA or FANCF loci targeted with d 20G- or e 21G-sgRNAs, measured by amplicon resequencing. f Bar chart of the total number of off-target sites detected by GUIDE-seq for SpCas9 variants on seven sites targeted with 20G-sgRNAs. a – e Tukey-type boxplots by BoxPlotR : center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the “minimum” and “maximum” data situated within 1.5 times the interquartile range from the 25th and 75th percentiles, respectively; notches indicate the 95% confidence intervals for the medians; crosses represent sample means; data points are plotted as open circles representing the mean of biologically independent triplicates. Spacers are schematically depicted beside the charts as combs: green color teeth indicate matching-, while a red color tooth indicates the presence of an appended nucleotide within the spacer; numbering of tooth position corresponds to the distance of the nucleotide from the PAM; the starting 20th nucleotide of the spacer is indicated by an uppercase letter and an appended 21st nucleotide by a red lowercase letter. See also Supplementary Figs. and .

Article Snippet: Plasmids developed by us and deposited at Addgene are the following: pX330-Flag-dSpCas9 (Addgene #92113), pX330-Flag-WT_SpCas9 (without sgRNA; with silent mutations) (Addgene #126753), pX330-Flag-eSpCas9 (without sgRNA; with silent mutations) (Addgene #126754), pX330-Flag-SpCas9-HF1 (without sgRNA; with silent mutations) (Addgene #126755), pX330-Flag-HypaSpCas9 (without sgRNA; with silent mutations) (Addgene #126756),pX330-Flag-evoSpCas9 (without sgRNA; with silent mutations) (Addgene #126758), pX330-Flag-HeFSpCas9 (without sgRNA; with silent mutations) (Addgene #126759), pX330-Flag-Sniper SpCas9 (without sgRNA; with silent mutations) (Addgene #126777), pX330-Flag-HiFi SpCas9 (without sgRNA; with silent mutations) (Addgene #126778), B-SpCas9 (Addgene #126760), B-eSpCas9 (Addgene #126761), B-SpCas9-HF1 (Addgene #126762), B-HypaSpCas9 (Addgene #126763), B-evoSpCas9 (Addgene #126765), B-HeFSpCas9 (Addgene #126766) eSpCas9-plus (Addgene #126767), SpCas9-HF1-plus (Addgene #126768) pET-FLAG-eSpCas9 (Addgene #126769), pET-FLAG-SpCas9-HF1 (Addgene #126770), pET-FLAG-B-eSpCas9 (Addgene #126772), pET-FLAG-eSpCas9-plus (Addgene #126774), pET-FLAG-SpCas9-HF1-plus (Addgene #126775) pmCherry_sgRNA-ver2 (Addgene #126776), pmCherry_gRNA (Addgene: #80457)

Techniques: Disruption, Activity Assay, Variant Assay, Amplification

a Pre-screening targets with increased fidelity nucleases for efficiency by the integration of a donor EGFP cassette. b Based on a the SpCas9-plus variants were selected to generate transgenic lines using the ‘self-cleaving’ EGFP-expression plasmid, which must integrate in-frame for Sprn promoter driven EGFP expression, and which downstream from the EGFP coding sequence it also contains a CMV-mCherry cassette; mCherry positive cells were counted. c Indel formation activity of eSpCas9-plus compared to WT and eSpCas9 with 21G-sgRNAs transcribed from an integrated single copy of lentiviruses measured by TIDE. a – c Means are shown, error bars represent the standard deviation (s.d.) for n = 3 biologically independent samples (overlaid as white circles). In the case of VEGFA site 8 targeted with WT and eSpCas9-plus on c one sample point is missing due to sample loss. Spacers are schematically depicted beside the charts as combs: green color teeth indicate matching-, while a red color tooth indicates the presence of an appended nucleotide within the spacer; numbering of tooth position corresponds to the distance of the nucleotide from the PAM; the starting 20th nucleotide of the spacer is indicated by an uppercase letter and an appended 21st nucleotide by a red lowercase letter.

Journal: Nature Communications

Article Title: Blackjack mutations improve the on-target activities of increased fidelity variants of SpCas9 with 5′G-extended sgRNAs

doi: 10.1038/s41467-020-15021-5

Figure Lengend Snippet: a Pre-screening targets with increased fidelity nucleases for efficiency by the integration of a donor EGFP cassette. b Based on a the SpCas9-plus variants were selected to generate transgenic lines using the ‘self-cleaving’ EGFP-expression plasmid, which must integrate in-frame for Sprn promoter driven EGFP expression, and which downstream from the EGFP coding sequence it also contains a CMV-mCherry cassette; mCherry positive cells were counted. c Indel formation activity of eSpCas9-plus compared to WT and eSpCas9 with 21G-sgRNAs transcribed from an integrated single copy of lentiviruses measured by TIDE. a – c Means are shown, error bars represent the standard deviation (s.d.) for n = 3 biologically independent samples (overlaid as white circles). In the case of VEGFA site 8 targeted with WT and eSpCas9-plus on c one sample point is missing due to sample loss. Spacers are schematically depicted beside the charts as combs: green color teeth indicate matching-, while a red color tooth indicates the presence of an appended nucleotide within the spacer; numbering of tooth position corresponds to the distance of the nucleotide from the PAM; the starting 20th nucleotide of the spacer is indicated by an uppercase letter and an appended 21st nucleotide by a red lowercase letter.

Article Snippet: Plasmids developed by us and deposited at Addgene are the following: pX330-Flag-dSpCas9 (Addgene #92113), pX330-Flag-WT_SpCas9 (without sgRNA; with silent mutations) (Addgene #126753), pX330-Flag-eSpCas9 (without sgRNA; with silent mutations) (Addgene #126754), pX330-Flag-SpCas9-HF1 (without sgRNA; with silent mutations) (Addgene #126755), pX330-Flag-HypaSpCas9 (without sgRNA; with silent mutations) (Addgene #126756),pX330-Flag-evoSpCas9 (without sgRNA; with silent mutations) (Addgene #126758), pX330-Flag-HeFSpCas9 (without sgRNA; with silent mutations) (Addgene #126759), pX330-Flag-Sniper SpCas9 (without sgRNA; with silent mutations) (Addgene #126777), pX330-Flag-HiFi SpCas9 (without sgRNA; with silent mutations) (Addgene #126778), B-SpCas9 (Addgene #126760), B-eSpCas9 (Addgene #126761), B-SpCas9-HF1 (Addgene #126762), B-HypaSpCas9 (Addgene #126763), B-evoSpCas9 (Addgene #126765), B-HeFSpCas9 (Addgene #126766) eSpCas9-plus (Addgene #126767), SpCas9-HF1-plus (Addgene #126768) pET-FLAG-eSpCas9 (Addgene #126769), pET-FLAG-SpCas9-HF1 (Addgene #126770), pET-FLAG-B-eSpCas9 (Addgene #126772), pET-FLAG-eSpCas9-plus (Addgene #126774), pET-FLAG-SpCas9-HF1-plus (Addgene #126775) pmCherry_sgRNA-ver2 (Addgene #126776), pmCherry_gRNA (Addgene: #80457)

Techniques: Transgenic Assay, Expressing, Plasmid Preparation, Sequencing, Activity Assay, Standard Deviation

A) Overall Structure of the Cas9-sgRNA-DNA Ternary Complex. Ribbon representation of the Cas9-sgRNA-DNA complex. Disordered linkers are shown as red dotted lines. Reproduced with permission from PMID: 24529477. B) 3-D Structure of the SpCas9 protein, showing the location of the identified immunodominant epitoes α and β. Reproduced with permission from PMID: 31015529.

Journal: Journal of pharmaceutical sciences

Article Title: Immunogenicity of Cas9 Protein

doi: 10.1016/j.xphs.2019.10.003

Figure Lengend Snippet: A) Overall Structure of the Cas9-sgRNA-DNA Ternary Complex. Ribbon representation of the Cas9-sgRNA-DNA complex. Disordered linkers are shown as red dotted lines. Reproduced with permission from PMID: 24529477. B) 3-D Structure of the SpCas9 protein, showing the location of the identified immunodominant epitoes α and β. Reproduced with permission from PMID: 31015529.

Article Snippet: Hereditary transthyretin amyloidosis , TTR , In vivo CRISPR–Cas9 gene editing to disrupt mutant TTR allele , NHEJ , In vivo lipid nanoparticle containing sgRNA and SpCas9 mRNA , Filing planned (2019) , Intellia Therapeutics, Regeneron Pharmaceuticals , Not indicated.

Techniques:

Clinical Trials involving CRISPR/Cas9

Journal: Journal of pharmaceutical sciences

Article Title: Immunogenicity of Cas9 Protein

doi: 10.1016/j.xphs.2019.10.003

Figure Lengend Snippet: Clinical Trials involving CRISPR/Cas9

Article Snippet: Hereditary transthyretin amyloidosis , TTR , In vivo CRISPR–Cas9 gene editing to disrupt mutant TTR allele , NHEJ , In vivo lipid nanoparticle containing sgRNA and SpCas9 mRNA , Filing planned (2019) , Intellia Therapeutics, Regeneron Pharmaceuticals , Not indicated.

Techniques: Clinical Proteomics, CRISPR, Ex Vivo, Electroporation, Modification, Injection, Knock-Out, In Vivo, Mutagenesis, Plasmid Preparation