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Targeted and genome-wide sequencing reveal single nucleotide variations impacting specificity of Cas9 in human stem cells.

Yang L, Grishin D, Wang G, Aach J, Zhang CZ, Chari R, Homsy J, Cai X, Zhao Y, Fan JB, Seidman C, Seidman J, Pu W, Church G - Nat Commun (2014)

Bottom Line: However, potential off-target effects of the Cas9 nuclease represent a major safety concern for any therapeutic application.We combine whole-genome sequencing and deep-targeted sequencing to characterise the off-target effects of Cas9 editing.Whole-genome sequencing of Cas9-modified hiPSC clones detects neither gross genomic alterations nor elevated mutation rates.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA [2] Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, USA.

ABSTRACT
CRISPR/Cas9 has demonstrated a high-efficiency in site-specific gene targeting. However, potential off-target effects of the Cas9 nuclease represent a major safety concern for any therapeutic application. Here, we knock out the Tafazzin gene by CRISPR/Cas9 in human-induced pluripotent stem cells with 54% efficiency. We combine whole-genome sequencing and deep-targeted sequencing to characterise the off-target effects of Cas9 editing. Whole-genome sequencing of Cas9-modified hiPSC clones detects neither gross genomic alterations nor elevated mutation rates. Deep sequencing of in silico predicted off-target sites in a population of Cas9-treated cells further confirms high specificity of Cas9. However, we identify a single high-efficiency off-target site that is generated by a common germline single-nucleotide variant (SNV) in our experiment. Based on in silico analysis, we estimate a likelihood of SNVs creating off-target sites in a human genome to be ~1.5-8.5%, depending on the genome and site-selection method, but also note that mutations might be generated at these sites only at low rates and may not have functional consequences. Our study demonstrates the feasibility of highly specific clonal ex vivo gene editing using CRISPR/Cas9 and highlights the value of whole-genome sequencing before personalised CRISPR design.

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Cas9 activity does not increase the rate of indels above background.(a) Frameshift deletions are introduced in the TAZ gene of two single-cell derived colonies TAZ1 and TAZ2; (b) Number of de novo indels in the control clone and TAZ1 and TAZ2 clones as detected by whole-genome sequencing analysis.
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f1: Cas9 activity does not increase the rate of indels above background.(a) Frameshift deletions are introduced in the TAZ gene of two single-cell derived colonies TAZ1 and TAZ2; (b) Number of de novo indels in the control clone and TAZ1 and TAZ2 clones as detected by whole-genome sequencing analysis.

Mentions: To target the TAZ gene, we integrated Cas9 under control of Tet-On transactivator into the genome of PGP1 (ref. 15) hiPSCs via the PiggyBac transposon and transfected the cells with a gRNA targeting a region (chr.X: 153,647,923–153,647,944) close to the mutation found in Barth Syndrome patients16. Genomic integration of Cas9 was chosen over transient transfection to ensure maximal Cas9 activity and enable high detection sensitivity. The control sample was transfected with a gRNA that has no similar site (≥15 bp of 20 bp) in hg19 reference genome (Supplementary Fig. 1). Single-cell derived hiPSC colonies were genotyped for on-target activity and the pluripotent state was verified through quantification of OCT4 and NANOG expression levels (Supplementary Figs 2 and 3). Two TAZ-knockout clones (TAZ1 and TAZ2) and one control clone were whole-genome sequenced. Short deletions at the target site were observed in both the TAZ-knockout clones as a result of Cas9 activity (Fig. 1a). No modification at the TAZ target site was observed in the control clone (Fig. 1a).


Targeted and genome-wide sequencing reveal single nucleotide variations impacting specificity of Cas9 in human stem cells.

Yang L, Grishin D, Wang G, Aach J, Zhang CZ, Chari R, Homsy J, Cai X, Zhao Y, Fan JB, Seidman C, Seidman J, Pu W, Church G - Nat Commun (2014)

Cas9 activity does not increase the rate of indels above background.(a) Frameshift deletions are introduced in the TAZ gene of two single-cell derived colonies TAZ1 and TAZ2; (b) Number of de novo indels in the control clone and TAZ1 and TAZ2 clones as detected by whole-genome sequencing analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352754&req=5

f1: Cas9 activity does not increase the rate of indels above background.(a) Frameshift deletions are introduced in the TAZ gene of two single-cell derived colonies TAZ1 and TAZ2; (b) Number of de novo indels in the control clone and TAZ1 and TAZ2 clones as detected by whole-genome sequencing analysis.
Mentions: To target the TAZ gene, we integrated Cas9 under control of Tet-On transactivator into the genome of PGP1 (ref. 15) hiPSCs via the PiggyBac transposon and transfected the cells with a gRNA targeting a region (chr.X: 153,647,923–153,647,944) close to the mutation found in Barth Syndrome patients16. Genomic integration of Cas9 was chosen over transient transfection to ensure maximal Cas9 activity and enable high detection sensitivity. The control sample was transfected with a gRNA that has no similar site (≥15 bp of 20 bp) in hg19 reference genome (Supplementary Fig. 1). Single-cell derived hiPSC colonies were genotyped for on-target activity and the pluripotent state was verified through quantification of OCT4 and NANOG expression levels (Supplementary Figs 2 and 3). Two TAZ-knockout clones (TAZ1 and TAZ2) and one control clone were whole-genome sequenced. Short deletions at the target site were observed in both the TAZ-knockout clones as a result of Cas9 activity (Fig. 1a). No modification at the TAZ target site was observed in the control clone (Fig. 1a).

Bottom Line: However, potential off-target effects of the Cas9 nuclease represent a major safety concern for any therapeutic application.We combine whole-genome sequencing and deep-targeted sequencing to characterise the off-target effects of Cas9 editing.Whole-genome sequencing of Cas9-modified hiPSC clones detects neither gross genomic alterations nor elevated mutation rates.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA [2] Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, USA.

ABSTRACT
CRISPR/Cas9 has demonstrated a high-efficiency in site-specific gene targeting. However, potential off-target effects of the Cas9 nuclease represent a major safety concern for any therapeutic application. Here, we knock out the Tafazzin gene by CRISPR/Cas9 in human-induced pluripotent stem cells with 54% efficiency. We combine whole-genome sequencing and deep-targeted sequencing to characterise the off-target effects of Cas9 editing. Whole-genome sequencing of Cas9-modified hiPSC clones detects neither gross genomic alterations nor elevated mutation rates. Deep sequencing of in silico predicted off-target sites in a population of Cas9-treated cells further confirms high specificity of Cas9. However, we identify a single high-efficiency off-target site that is generated by a common germline single-nucleotide variant (SNV) in our experiment. Based on in silico analysis, we estimate a likelihood of SNVs creating off-target sites in a human genome to be ~1.5-8.5%, depending on the genome and site-selection method, but also note that mutations might be generated at these sites only at low rates and may not have functional consequences. Our study demonstrates the feasibility of highly specific clonal ex vivo gene editing using CRISPR/Cas9 and highlights the value of whole-genome sequencing before personalised CRISPR design.

Show MeSH