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CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.

Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM - Nat. Biotechnol. (2013)

Bottom Line: Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1-3 and 1-2 target mismatches, respectively.By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena.Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. [2].

ABSTRACT
Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease- Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a transcriptional activation-based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator-like (TALs) effectors. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1-3 and 1-2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.

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Off-set nicking(a) We employed the traffic light reporter29 to simultaneously assay for HR and NHEJevents upon introduction of targeted nicks or breaks: DNA cleavage eventsresolved through the HDR pathway restore the GFP sequence (via a donortemplate), whereas mutagenic NHEJ causes frame-shifts rendering the GFP out offrame and the downstream mCherry sequence in frame. For the assay, we designed14 sgRNAs covering a 200bp stretch of DNA: 7 targeting the sense strand(U1–7) and 7 the antisense strand (D1–7). Using the Cas9D10Amutant, which nicks the complementary strand, we used different two-waycombinations of the sgRNAs to induce a range of programmed 5′ or3′ overhangs (the nicking sites for the 14 sgRNAs are indicated).(b) Inducing off-set nicks to generate DSBs is highly effectiveat inducing gene disruption. Notably off-set nicks leading to 5′overhangs result in more NHEJ events as opposed to 3′ overhangs.(c) Again, off-set nicks leading to 5′ overhangs alsoresult in more HR and NHEJ events as opposed to 3′ overhangs. In(b,c) the predicted overhang lengths are indicated below thecorresponding x-axis legends. Data are means +/− SEM(N=3).
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Figure 3: Off-set nicking(a) We employed the traffic light reporter29 to simultaneously assay for HR and NHEJevents upon introduction of targeted nicks or breaks: DNA cleavage eventsresolved through the HDR pathway restore the GFP sequence (via a donortemplate), whereas mutagenic NHEJ causes frame-shifts rendering the GFP out offrame and the downstream mCherry sequence in frame. For the assay, we designed14 sgRNAs covering a 200bp stretch of DNA: 7 targeting the sense strand(U1–7) and 7 the antisense strand (D1–7). Using the Cas9D10Amutant, which nicks the complementary strand, we used different two-waycombinations of the sgRNAs to induce a range of programmed 5′ or3′ overhangs (the nicking sites for the 14 sgRNAs are indicated).(b) Inducing off-set nicks to generate DSBs is highly effectiveat inducing gene disruption. Notably off-set nicks leading to 5′overhangs result in more NHEJ events as opposed to 3′ overhangs.(c) Again, off-set nicks leading to 5′ overhangs alsoresult in more HR and NHEJ events as opposed to 3′ overhangs. In(b,c) the predicted overhang lengths are indicated below thecorresponding x-axis legends. Data are means +/− SEM(N=3).

Mentions: In the context of genome-editing, we chose to focus on creating off-set nicks togenerate DSBs. Our motivation stems from the observation (Supplementary Fig. 15) that a largemajority of nicks do not result in non-homologous end joining (NHEJ) mediatedindels29., and thus wheninducing off-set nicks, off-target single nick events will likely result in very lowindel rates. Towards this we found that inducing off-set nicks to generate DSBs ishighly effective at inducing gene disruption at both integrated reporter loci (Fig. 3) and at the native AAVS1 genomic locus (Supplementary Figs. 16, 17).Furthermore, we also noted that consistent with the standard model for homologousrecombination (HR) mediated repair30engineering of 5′ overhangs via off-set nicks generated more robust NHEJ eventsthan 3′ overhangs (Fig. 3b). In addition toa stimulation of NHEJ, we also observed robust induction of HR when the 5′overhangs were created. generation of 3′ overhangs did not result in improvementof HR rates (Figs. 3c). It remains to be determinedif Cas9 biochemistry or chromatin state and nucleotide composition of the genomic locialso contributed to the observed results above. While we did not actually measureoff-target activity of this methodology, we believe the use of cooperativity such aswith off-set nicks for generating DSBs offers a promising route for mitigating theeffects of off-target sgRNA:Cas9 activity.


CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.

Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM - Nat. Biotechnol. (2013)

Off-set nicking(a) We employed the traffic light reporter29 to simultaneously assay for HR and NHEJevents upon introduction of targeted nicks or breaks: DNA cleavage eventsresolved through the HDR pathway restore the GFP sequence (via a donortemplate), whereas mutagenic NHEJ causes frame-shifts rendering the GFP out offrame and the downstream mCherry sequence in frame. For the assay, we designed14 sgRNAs covering a 200bp stretch of DNA: 7 targeting the sense strand(U1–7) and 7 the antisense strand (D1–7). Using the Cas9D10Amutant, which nicks the complementary strand, we used different two-waycombinations of the sgRNAs to induce a range of programmed 5′ or3′ overhangs (the nicking sites for the 14 sgRNAs are indicated).(b) Inducing off-set nicks to generate DSBs is highly effectiveat inducing gene disruption. Notably off-set nicks leading to 5′overhangs result in more NHEJ events as opposed to 3′ overhangs.(c) Again, off-set nicks leading to 5′ overhangs alsoresult in more HR and NHEJ events as opposed to 3′ overhangs. In(b,c) the predicted overhang lengths are indicated below thecorresponding x-axis legends. Data are means +/− SEM(N=3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3818127&req=5

Figure 3: Off-set nicking(a) We employed the traffic light reporter29 to simultaneously assay for HR and NHEJevents upon introduction of targeted nicks or breaks: DNA cleavage eventsresolved through the HDR pathway restore the GFP sequence (via a donortemplate), whereas mutagenic NHEJ causes frame-shifts rendering the GFP out offrame and the downstream mCherry sequence in frame. For the assay, we designed14 sgRNAs covering a 200bp stretch of DNA: 7 targeting the sense strand(U1–7) and 7 the antisense strand (D1–7). Using the Cas9D10Amutant, which nicks the complementary strand, we used different two-waycombinations of the sgRNAs to induce a range of programmed 5′ or3′ overhangs (the nicking sites for the 14 sgRNAs are indicated).(b) Inducing off-set nicks to generate DSBs is highly effectiveat inducing gene disruption. Notably off-set nicks leading to 5′overhangs result in more NHEJ events as opposed to 3′ overhangs.(c) Again, off-set nicks leading to 5′ overhangs alsoresult in more HR and NHEJ events as opposed to 3′ overhangs. In(b,c) the predicted overhang lengths are indicated below thecorresponding x-axis legends. Data are means +/− SEM(N=3).
Mentions: In the context of genome-editing, we chose to focus on creating off-set nicks togenerate DSBs. Our motivation stems from the observation (Supplementary Fig. 15) that a largemajority of nicks do not result in non-homologous end joining (NHEJ) mediatedindels29., and thus wheninducing off-set nicks, off-target single nick events will likely result in very lowindel rates. Towards this we found that inducing off-set nicks to generate DSBs ishighly effective at inducing gene disruption at both integrated reporter loci (Fig. 3) and at the native AAVS1 genomic locus (Supplementary Figs. 16, 17).Furthermore, we also noted that consistent with the standard model for homologousrecombination (HR) mediated repair30engineering of 5′ overhangs via off-set nicks generated more robust NHEJ eventsthan 3′ overhangs (Fig. 3b). In addition toa stimulation of NHEJ, we also observed robust induction of HR when the 5′overhangs were created. generation of 3′ overhangs did not result in improvementof HR rates (Figs. 3c). It remains to be determinedif Cas9 biochemistry or chromatin state and nucleotide composition of the genomic locialso contributed to the observed results above. While we did not actually measureoff-target activity of this methodology, we believe the use of cooperativity such aswith off-set nicks for generating DSBs offers a promising route for mitigating theeffects of off-target sgRNA:Cas9 activity.

Bottom Line: Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1-3 and 1-2 target mismatches, respectively.By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena.Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. [2].

ABSTRACT
Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease- Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a transcriptional activation-based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator-like (TALs) effectors. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1-3 and 1-2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.

Show MeSH
Related in: MedlinePlus