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119. Engineering a Self-Inactivating CRISPR System for AAV Vectors
Targeted Genome Editing I 119. Engineering a Self-Inactivating CRISPR System for AAV Vectors
Benjamin E. Epstein, David V. Schaffer Bioengineering, University of California, Berkeley, Berkeley, CA Significant strides in nuclease engineering have enabled a broad range of biomedical applications. However, numerous challenges remain, and in particular the long term expression mediated by AAV delivery of nuclease-encoding constructs to post-mitotic cells raises concerns with specificity and immunogenicity. That is, off-target nuclease activity can induce genotoxicity, and expression of an exogenous nuclease has the potential to elicit an immune response against transduced cells. Thus, it would be advantageous to limit the duration of nuclease expression following delivery. We have engineered self-inactivating nuclease constructs using the CRISPR/Cas9 system, which consist of a Cas9 nuclease such as that from Streptococcus pyogenes (SpCas9), a chimeric single guide RNA (sgRNA) molecule for targeting, and flanking sites targeted by that sgRNA. For example, we modified an SpCas9/sgRNA construct targeted at the VEGFA locus by introducing copies of the target site flanking the nuclease construct. The result is a negative feedback loop where Cas9 cuts both the target genomic locus and its own coding construct and thereby self-limits its expression. We demonstrate that this construct can eliminate >90% of its expression within 72 hours, and tuning of different parameters enables retention of up to 65% on-target efficiency and reduction of off target-cutting by up to 80%. We further show that by engineering the flanking target sites to contain mismatches to the sgRNA, the Cas9 expression duration can be modulated and the on-target/off-target cutting ratios improved. By retaining strong activity while minimizing off-target effects and by eliminating potentially immunogenic, long-term expression of foreign protein, these self-inactivating constructs have the potential to address two substantial concerns with therapeutic application of engineered nucleases.
120. Efficient In Vivo Liver-Directed Gene Editing Using CRISPR/Cas9
Kshitiz Singh, Hanneke Evens, Melvin Rincón, Nisha Nair, Shilpita Sarcar, Ermira Samara-Kuko, Marinee K. Chuah, Thierry VandenDriessche Department of Gene Therapy & Regenerative Medicine, Free University of Brussels, Brussels, Belgium
Clustered, regularly interspersed, short palindromic repeat RNAguided nucleases have emerged as highly efficient genome editing tools. However, in vivo tissue-specific genome editing at the desired loci is still a challenge. Here, we report that truncated guide RNAs (gRNAs) and Cas9 under the control of a computationally designed hepatocyte-specific promoter lead to liver-specific and target sitespecific indel formation in the mouse factor IX (FIX) gene. The truncated gRNAs targeting unique sites in exon 1 and exon 6 of the mouse FIX gene were designed using a computational CRISPR design tool and the target sites overlap with mutations known to cause hemophilia B in patients. The gRNA and Cas9-expressing constructs were delivered in vivo using AAV9 vectors. The efficiency of in vivo targeting was assessed by T7E1 assays, site-specific Sanger sequencing and deep sequencing of on-target and putative off-target sites. Though AAV9 transduction was apparent in multiple tissues and organs, Cas9 expression was restricted mainly to the liver, with only minimal or no expression in other non-hepatic tissues. Consequently, the indel frequency was robust in the liver (up to 50%) in the desired target locus of the FIX gene, with no evidence of targeting in other organs. This resulted in a substantial loss of FIX activity and the emergence of a bleeding phenotype, consistent with hemophilia B.
S50
Deep sequencing of putative off-target sites revealed no off-target editing. Our findings have potentially broad implications for somatic gene editing in the liver using the CRISPR/Cas9 platform.
121. T Cell Receptor Modification by Highly Specific TALEN and CRISPR/Cas9
Friederike Knipping1, Karl Petri1, Eliana Ruggiero1, Mark Osborn2, Jakub Tolar2, Manfred Schmidt1, Christof von Kalle1, Richard Gabriel1 1 Department of Translational Oncology, German Cancer Research Center and National Center for Tumor Diseases, Heidelberg, Germany, 2Department of Pediatrics, University of Minnesota, Minneapolis, MN Adoptive transfer of T cells with transgenic high avidity T cell receptors (TCR) is a promising therapeutic approach, however it comprises certain challenges. Endogenous and transferred TCR chains compete for surface expression and may pair inappropriately, potentially leading to autoimmunity. This can be prevented by designer nucleases such as transcription activator-like effector nucleases (TALEN) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) system. Their ability to introduce specific DNA double strand breaks (DSB) at their target sites can be harnessed for targeted genome editing. DSB repair through non-homologous end joining (NHEJ) can result in permanent gene knockout due to frame shift mutations. Alternatively, if a homology-containing donor template is provided, transgenes can be integrated into the specific target locus by homology directed repair (HDR). In order to disrupt endogenous TCR expression, we assembled nine TALEN and two CRISPR/Cas9 guide RNA (gRNA) targeting the constant region of the TCR α-chain (TRAC) and four TALEN and three CRISPR/Cas9 gRNA targeting both constant regions of the TCR β-chain (TRBC1/TRBC2). Here we show specific DSB induction by TALEN and CRISPR/Cas9 in K562 and primary T cells using T7 endonuclease I targeting efficiency assay and deep sequencing. Electroporation of primary T cells with TRAC-TALEN or TRBC-TALEN mRNA led to successful elimination of surface TCR expression in about 76% of the cells. To analyze nuclease specificity, K562 cells were transduced with an integrase-defective lentiviral vector (IDLV) prior to nucleofection with TALEN- or CRISPR/Cas9expressing plasmids. IDLV can be integrated into DSB during DNA repair, thereby serving as stable markers for transient DSB. IDLVmarked DSB were subsequently localized using LAM-PCR and deep sequencing. Clustered integration sites (CLIS) were detected around all nuclease target sites, confirming on-target activity. Although up to 3268 IDLV integration sites were analyzed for each TALEN and CRISPR/Cas9 gRNA, only one CLIS for one gRNA was mapped at an off-target position, indicating a very high level of specificity. To establish HDR-mediated targeted integration, we assembled donor templates containing a GFP expression cassette flanked by 800bp TRAC- or TRBC1-homologous sequences. We verified targeted gene addition in 10% of K562 cells treated with TALEN and the respective donor template. Delivery of the TRAC-donor packaged into IDLV resulted in targeted integration of the GFP expression cassette in 5% of primary T cells. To sum up, here we present highly efficient and specific TALEN and CRISPR/Cas9 and their utility for T cell engineering.
Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
Benjamin E. Epstein, David V. Schaffer Bioengineering, University of California, Berkeley, Berkeley, CA Significant strides in nuclease engineering have enabled a broad range of biomedical applications. However, numerous challenges remain, and in particular the long term expression mediated by AAV delivery of nuclease-encoding constructs to post-mitotic cells raises concerns with specificity and immunogenicity. That is, off-target nuclease activity can induce genotoxicity, and expression of an exogenous nuclease has the potential to elicit an immune response against transduced cells. Thus, it would be advantageous to limit the duration of nuclease expression following delivery. We have engineered self-inactivating nuclease constructs using the CRISPR/Cas9 system, which consist of a Cas9 nuclease such as that from Streptococcus pyogenes (SpCas9), a chimeric single guide RNA (sgRNA) molecule for targeting, and flanking sites targeted by that sgRNA. For example, we modified an SpCas9/sgRNA construct targeted at the VEGFA locus by introducing copies of the target site flanking the nuclease construct. The result is a negative feedback loop where Cas9 cuts both the target genomic locus and its own coding construct and thereby self-limits its expression. We demonstrate that this construct can eliminate >90% of its expression within 72 hours, and tuning of different parameters enables retention of up to 65% on-target efficiency and reduction of off target-cutting by up to 80%. We further show that by engineering the flanking target sites to contain mismatches to the sgRNA, the Cas9 expression duration can be modulated and the on-target/off-target cutting ratios improved. By retaining strong activity while minimizing off-target effects and by eliminating potentially immunogenic, long-term expression of foreign protein, these self-inactivating constructs have the potential to address two substantial concerns with therapeutic application of engineered nucleases.
120. Efficient In Vivo Liver-Directed Gene Editing Using CRISPR/Cas9
Kshitiz Singh, Hanneke Evens, Melvin Rincón, Nisha Nair, Shilpita Sarcar, Ermira Samara-Kuko, Marinee K. Chuah, Thierry VandenDriessche Department of Gene Therapy & Regenerative Medicine, Free University of Brussels, Brussels, Belgium
Clustered, regularly interspersed, short palindromic repeat RNAguided nucleases have emerged as highly efficient genome editing tools. However, in vivo tissue-specific genome editing at the desired loci is still a challenge. Here, we report that truncated guide RNAs (gRNAs) and Cas9 under the control of a computationally designed hepatocyte-specific promoter lead to liver-specific and target sitespecific indel formation in the mouse factor IX (FIX) gene. The truncated gRNAs targeting unique sites in exon 1 and exon 6 of the mouse FIX gene were designed using a computational CRISPR design tool and the target sites overlap with mutations known to cause hemophilia B in patients. The gRNA and Cas9-expressing constructs were delivered in vivo using AAV9 vectors. The efficiency of in vivo targeting was assessed by T7E1 assays, site-specific Sanger sequencing and deep sequencing of on-target and putative off-target sites. Though AAV9 transduction was apparent in multiple tissues and organs, Cas9 expression was restricted mainly to the liver, with only minimal or no expression in other non-hepatic tissues. Consequently, the indel frequency was robust in the liver (up to 50%) in the desired target locus of the FIX gene, with no evidence of targeting in other organs. This resulted in a substantial loss of FIX activity and the emergence of a bleeding phenotype, consistent with hemophilia B.
S50
Deep sequencing of putative off-target sites revealed no off-target editing. Our findings have potentially broad implications for somatic gene editing in the liver using the CRISPR/Cas9 platform.
121. T Cell Receptor Modification by Highly Specific TALEN and CRISPR/Cas9
Friederike Knipping1, Karl Petri1, Eliana Ruggiero1, Mark Osborn2, Jakub Tolar2, Manfred Schmidt1, Christof von Kalle1, Richard Gabriel1 1 Department of Translational Oncology, German Cancer Research Center and National Center for Tumor Diseases, Heidelberg, Germany, 2Department of Pediatrics, University of Minnesota, Minneapolis, MN Adoptive transfer of T cells with transgenic high avidity T cell receptors (TCR) is a promising therapeutic approach, however it comprises certain challenges. Endogenous and transferred TCR chains compete for surface expression and may pair inappropriately, potentially leading to autoimmunity. This can be prevented by designer nucleases such as transcription activator-like effector nucleases (TALEN) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) system. Their ability to introduce specific DNA double strand breaks (DSB) at their target sites can be harnessed for targeted genome editing. DSB repair through non-homologous end joining (NHEJ) can result in permanent gene knockout due to frame shift mutations. Alternatively, if a homology-containing donor template is provided, transgenes can be integrated into the specific target locus by homology directed repair (HDR). In order to disrupt endogenous TCR expression, we assembled nine TALEN and two CRISPR/Cas9 guide RNA (gRNA) targeting the constant region of the TCR α-chain (TRAC) and four TALEN and three CRISPR/Cas9 gRNA targeting both constant regions of the TCR β-chain (TRBC1/TRBC2). Here we show specific DSB induction by TALEN and CRISPR/Cas9 in K562 and primary T cells using T7 endonuclease I targeting efficiency assay and deep sequencing. Electroporation of primary T cells with TRAC-TALEN or TRBC-TALEN mRNA led to successful elimination of surface TCR expression in about 76% of the cells. To analyze nuclease specificity, K562 cells were transduced with an integrase-defective lentiviral vector (IDLV) prior to nucleofection with TALEN- or CRISPR/Cas9expressing plasmids. IDLV can be integrated into DSB during DNA repair, thereby serving as stable markers for transient DSB. IDLVmarked DSB were subsequently localized using LAM-PCR and deep sequencing. Clustered integration sites (CLIS) were detected around all nuclease target sites, confirming on-target activity. Although up to 3268 IDLV integration sites were analyzed for each TALEN and CRISPR/Cas9 gRNA, only one CLIS for one gRNA was mapped at an off-target position, indicating a very high level of specificity. To establish HDR-mediated targeted integration, we assembled donor templates containing a GFP expression cassette flanked by 800bp TRAC- or TRBC1-homologous sequences. We verified targeted gene addition in 10% of K562 cells treated with TALEN and the respective donor template. Delivery of the TRAC-donor packaged into IDLV resulted in targeted integration of the GFP expression cassette in 5% of primary T cells. To sum up, here we present highly efficient and specific TALEN and CRISPR/Cas9 and their utility for T cell engineering.
Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy