Wild-type Cas9 Research Articles

All-in-One CRISPR-Cas9/FokI-dCas9 Vector-Mediated Multiplex Genome Engineering in Cultured Cells.

CRISPR-Cas9 enables highly convenient multiplex genome engineering in cultured cells, because it utilizes generic Cas9 nuclease and an easily customizable single-guide RNA (sgRNA) for site-specific DNA double-strand break induction. We previously established a multiplex CRISPR-Cas9 assembly system for constructing an all-in-one vector simultaneously expressing multiple sgRNAs and Cas9 nuclease or other Cas9 variants including FokI-dCas9, which supersedes the wild-type Cas9 with regard to high specificity. In this chapter, we describe a streamlined protocol to design and construct multiplex CRISPR-Cas9 or FokI-dCas9 vectors, to introduce them into cultured cells by lipofection or electroporation, to enrich the genomically edited cells with a transient puromycin selection, to validate the mutation efficiency by Surveyor nuclease assay, and to perform off-target analyses. We show that our protocol enables highly efficient multiplex genome engineering even in hard-to-transfect HepG2 cells.

The Convenience of Single Homology Arm Donor DNA and CRISPR/Cas9-Nickase for Targeted Insertion of Long DNA Fragment.

CRISPR/Cas9 technology provides a powerful tool for targeted modification of genomes. In this system, a donor DNA harboring two flanking homology arms is mostly used for targeted insertion of long exogenous DNA. Here, we introduced an alternative design for the donor DNA by incorporation of a single short homology arm into a circular plasmid. In this experimental study, single homology arm donor was applied along with a single guide RNA (sgRNA) specific to the homology region, and either Cas9 or its mutant nickase variant (Cas9n). Using Pdx1 gene as the target locus the functionality of this system was evaluated in MIN6 cell line and murine embryonic stem cells (ESCs). Both wild type Cas9 and Cas9n could conduct the knock-in process with this system. We successfully applied this strategy with Cas9n for generation of Pdx1GFP knock-in mouse ESC lines. Altogether, our results demonstrated that a combination of a single homology arm donor, a single guide RNA and Cas9n is capable of precisely incorporating DNA fragments of multiple kilo base pairs into the targeted genomic locus. While taking advantage of low off-target mutagenesis of the Cas9n, our new design strategy may facilitate the targeting process. Consequently, this strategy can be applied in knock-in or insertional inactivation studies.

167 Long-term in vivo correction of a recessive dystrophic epidermolysis bullosa phenotype using RNA trans-splicing repair

164 CRISPR/Cas9-mediated gene repair in the COL7A1 gene S Hainzl, T Kocher, EM Murauer, F Larcher, M Steiner, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria, 2 Department of Dermatology, Paracelsus Medical University, Salzburg, Austria, 3 Epithelial Biomedicine Division, CIEMAT, Madrid, Spain and 4 3rd Medical Department, Paracelsus Medical University Salzburg, Austria, Laboratory for Immunological and Molecular Cancer Research, Salzburg, Austria The CRISPR/Cas9 system turned out to be a powerful tool for genome editing and is therefore a promising option for the specific repair of gene mutations causing the blistering skin disease epidermolysis bullosa (EB). We have exploited the CRISPR/Cas9-mediated homologydirected repair (HDR) approach for the correction of a homozygous mutation in COL7A1 exon 80, leading to a complete loss of type VII collagen within the basement membrane zone of the skin. We have predicted a guide RNA (gRNA) specific for intron 80 of COL7A1, which was then cloned either into a wild-type Cas9 dual vector system, inducing double strand breaks, or a D10A Cas9 dual vector system, causing single strand breaks within the target intron. Homology COL7A1 arms for HDR were cloned into a donor vector, including a selection cassette. Transfected patient keratinocytes were selected either via antibiotic selection or fluorescent-activated cell sorting (FACS). RT-PCR on genomic DNA of treated cells and subsequent restriction enzyme digest analysis of the resulting PCR products showed the genetic correction of the COL7A1 mutation. The mutation-specific enzymatic digest revealed the presence of 26% reverted alleles. Additionally, type VII collagen restoration was confirmed via Western blot analysis and immunofluorescence staining. Our data indicate that genome editing using the CRISPR/Cas9 system can be an elegant tool for the repair of genes involved in the severe skin disease epidermolysis bullosa. 165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa B Liemberger, C Arzt, S Hainzl, J Pinon Hofbauer, V Wally, EM Murauer, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria and 2 Department of Dermatology, Paracelsus Medical University Salzburg, Austria, Salzburg, Austria Epidermolysis bullosa (EB) is an inherited skin disease characterized by severe blistering of the skin and mucous membranes after minor mechanical trauma. In EB simplex (EBS) dominant mutations within KRT14 encoding keratin 14 lead to loss of integrity of the intermediate filament network within basal keratinocytes. In order to correct a dominant mutation in an EBS patient cell line, we exploited the RNA trans-splicing technology which utilizes the cell’s endogenous splicing machinery to facilitate a trans-splicing reaction between two RNAs, generating a new chimeric product at the pre-mRNA level. The designed RNA transsplicing molecule (RTM), containing a binding domain, splicing elements, and the wild-type KRT14 region to be introduced, has already been shown to be able to partially revert the EBS phenotype at the cellular level. In order to increase the repair efficiency mediated by RNA trans-splicing, antisense RNAs (asRNAs) specific for splice sites or splicing enhancer sequences within the KRT14 target pre-mRNA region were randomly generated with the aim of blocking these cis-splicing elements thereby increasing the trans-splicing rate. We analysed the functionality of 76 individual asRNAs by exploiting our fluorescence-based screening system, in which reconstitution of a GFP signal acts as a readout for accurate trans-splicing between a generated KRT14 minigene (KRT14-MG) and the RTM. Triple transfection experiments in HEK293 cells of the KRT14-MG, the RTM, and the most functional asRNA revealed an increase in trans-splicing efficiency of up to 7 fold as monitored by flow cytometry. Our data suggest that the inclusion of asRNAs can increase the trans-splicing efficiency to levels needed to overcome the EBS phenotype.

164 CRISPR/Cas9-mediated gene repair in the COL7A1 gene

The CRISPR/Cas9 system turned out to be a powerful tool for genome editing and is therefore a promising option for the specific repair of gene mutations causing the blistering skin disease epidermolysis bullosa (EB). We have exploited the CRISPR/Cas9-mediated homology-directed repair (HDR) approach for the correction of a homozygous mutation in COL7A1 exon 80, leading to a complete loss of type VII collagen within the basement membrane zone of the skin. We have predicted a guide RNA (gRNA) specific for intron 80 of COL7A1, which was then cloned either into a wild-type Cas9 dual vector system, inducing double strand breaks, or a D10A Cas9 dual vector system, causing single strand breaks within the target intron. Homology COL7A1 arms for HDR were cloned into a donor vector, including a selection cassette. Transfected patient keratinocytes were selected either via antibiotic selection or fluorescent-activated cell sorting (FACS). RT-PCR on genomic DNA of treated cells and subsequent restriction enzyme digest analysis of the resulting PCR products showed the genetic correction of the COL7A1 mutation. The mutation-specific enzymatic digest revealed the presence of 26% reverted alleles. Additionally, type VII collagen restoration was confirmed via Western blot analysis and immunofluorescence staining. Our data indicate that genome editing using the CRISPR/Cas9 system can be an elegant tool for the repair of genes involved in the severe skin disease epidermolysis bullosa.

165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa

164 CRISPR/Cas9-mediated gene repair in the COL7A1 gene S Hainzl, T Kocher, EM Murauer, F Larcher, M Steiner, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria, 2 Department of Dermatology, Paracelsus Medical University, Salzburg, Austria, 3 Epithelial Biomedicine Division, CIEMAT, Madrid, Spain and 4 3rd Medical Department, Paracelsus Medical University Salzburg, Austria, Laboratory for Immunological and Molecular Cancer Research, Salzburg, Austria The CRISPR/Cas9 system turned out to be a powerful tool for genome editing and is therefore a promising option for the specific repair of gene mutations causing the blistering skin disease epidermolysis bullosa (EB). We have exploited the CRISPR/Cas9-mediated homologydirected repair (HDR) approach for the correction of a homozygous mutation in COL7A1 exon 80, leading to a complete loss of type VII collagen within the basement membrane zone of the skin. We have predicted a guide RNA (gRNA) specific for intron 80 of COL7A1, which was then cloned either into a wild-type Cas9 dual vector system, inducing double strand breaks, or a D10A Cas9 dual vector system, causing single strand breaks within the target intron. Homology COL7A1 arms for HDR were cloned into a donor vector, including a selection cassette. Transfected patient keratinocytes were selected either via antibiotic selection or fluorescent-activated cell sorting (FACS). RT-PCR on genomic DNA of treated cells and subsequent restriction enzyme digest analysis of the resulting PCR products showed the genetic correction of the COL7A1 mutation. The mutation-specific enzymatic digest revealed the presence of 26% reverted alleles. Additionally, type VII collagen restoration was confirmed via Western blot analysis and immunofluorescence staining. Our data indicate that genome editing using the CRISPR/Cas9 system can be an elegant tool for the repair of genes involved in the severe skin disease epidermolysis bullosa. 165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa B Liemberger, C Arzt, S Hainzl, J Pinon Hofbauer, V Wally, EM Murauer, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria and 2 Department of Dermatology, Paracelsus Medical University Salzburg, Austria, Salzburg, Austria Epidermolysis bullosa (EB) is an inherited skin disease characterized by severe blistering of the skin and mucous membranes after minor mechanical trauma. In EB simplex (EBS) dominant mutations within KRT14 encoding keratin 14 lead to loss of integrity of the intermediate filament network within basal keratinocytes. In order to correct a dominant mutation in an EBS patient cell line, we exploited the RNA trans-splicing technology which utilizes the cell’s endogenous splicing machinery to facilitate a trans-splicing reaction between two RNAs, generating a new chimeric product at the pre-mRNA level. The designed RNA transsplicing molecule (RTM), containing a binding domain, splicing elements, and the wild-type KRT14 region to be introduced, has already been shown to be able to partially revert the EBS phenotype at the cellular level. In order to increase the repair efficiency mediated by RNA trans-splicing, antisense RNAs (asRNAs) specific for splice sites or splicing enhancer sequences within the KRT14 target pre-mRNA region were randomly generated with the aim of blocking these cis-splicing elements thereby increasing the trans-splicing rate. We analysed the functionality of 76 individual asRNAs by exploiting our fluorescence-based screening system, in which reconstitution of a GFP signal acts as a readout for accurate trans-splicing between a generated KRT14 minigene (KRT14-MG) and the RTM. Triple transfection experiments in HEK293 cells of the KRT14-MG, the RTM, and the most functional asRNA revealed an increase in trans-splicing efficiency of up to 7 fold as monitored by flow cytometry. Our data suggest that the inclusion of asRNAs can increase the trans-splicing efficiency to levels needed to overcome the EBS phenotype.

169 Regeneration of a functional epidermis at a large, long-standing wound by gene-corrected autologous epidermal stem cells

164 CRISPR/Cas9-mediated gene repair in the COL7A1 gene S Hainzl, T Kocher, EM Murauer, F Larcher, M Steiner, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria, 2 Department of Dermatology, Paracelsus Medical University, Salzburg, Austria, 3 Epithelial Biomedicine Division, CIEMAT, Madrid, Spain and 4 3rd Medical Department, Paracelsus Medical University Salzburg, Austria, Laboratory for Immunological and Molecular Cancer Research, Salzburg, Austria The CRISPR/Cas9 system turned out to be a powerful tool for genome editing and is therefore a promising option for the specific repair of gene mutations causing the blistering skin disease epidermolysis bullosa (EB). We have exploited the CRISPR/Cas9-mediated homologydirected repair (HDR) approach for the correction of a homozygous mutation in COL7A1 exon 80, leading to a complete loss of type VII collagen within the basement membrane zone of the skin. We have predicted a guide RNA (gRNA) specific for intron 80 of COL7A1, which was then cloned either into a wild-type Cas9 dual vector system, inducing double strand breaks, or a D10A Cas9 dual vector system, causing single strand breaks within the target intron. Homology COL7A1 arms for HDR were cloned into a donor vector, including a selection cassette. Transfected patient keratinocytes were selected either via antibiotic selection or fluorescent-activated cell sorting (FACS). RT-PCR on genomic DNA of treated cells and subsequent restriction enzyme digest analysis of the resulting PCR products showed the genetic correction of the COL7A1 mutation. The mutation-specific enzymatic digest revealed the presence of 26% reverted alleles. Additionally, type VII collagen restoration was confirmed via Western blot analysis and immunofluorescence staining. Our data indicate that genome editing using the CRISPR/Cas9 system can be an elegant tool for the repair of genes involved in the severe skin disease epidermolysis bullosa. 165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa B Liemberger, C Arzt, S Hainzl, J Pinon Hofbauer, V Wally, EM Murauer, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria and 2 Department of Dermatology, Paracelsus Medical University Salzburg, Austria, Salzburg, Austria Epidermolysis bullosa (EB) is an inherited skin disease characterized by severe blistering of the skin and mucous membranes after minor mechanical trauma. In EB simplex (EBS) dominant mutations within KRT14 encoding keratin 14 lead to loss of integrity of the intermediate filament network within basal keratinocytes. In order to correct a dominant mutation in an EBS patient cell line, we exploited the RNA trans-splicing technology which utilizes the cell’s endogenous splicing machinery to facilitate a trans-splicing reaction between two RNAs, generating a new chimeric product at the pre-mRNA level. The designed RNA transsplicing molecule (RTM), containing a binding domain, splicing elements, and the wild-type KRT14 region to be introduced, has already been shown to be able to partially revert the EBS phenotype at the cellular level. In order to increase the repair efficiency mediated by RNA trans-splicing, antisense RNAs (asRNAs) specific for splice sites or splicing enhancer sequences within the KRT14 target pre-mRNA region were randomly generated with the aim of blocking these cis-splicing elements thereby increasing the trans-splicing rate. We analysed the functionality of 76 individual asRNAs by exploiting our fluorescence-based screening system, in which reconstitution of a GFP signal acts as a readout for accurate trans-splicing between a generated KRT14 minigene (KRT14-MG) and the RTM. Triple transfection experiments in HEK293 cells of the KRT14-MG, the RTM, and the most functional asRNA revealed an increase in trans-splicing efficiency of up to 7 fold as monitored by flow cytometry. Our data suggest that the inclusion of asRNAs can increase the trans-splicing efficiency to levels needed to overcome the EBS phenotype.

Abstract 71: Cas9 protein engineering for cell cycle-specific genome editing to enhance homology directed repair

Abstract The comprehensive and coordinated efforts of The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) identified the somatic genomic alterations occurring in a broad panel of human cancers. The challenge ahead is to understand the functions of these somatically altered genes.Genome editing tools like the CRISPR/Cas9 system represent a transformative technology that allows the precise engineering of cells harboring specific (point) mutations found in specific tumor subtypes, enabling the investigation of the role of a candidate cancer gene in a stringently defined genetic context. Once recruited to its DNA target sequence in cells, Cas9 induces a DNA double strand break (DSB), which is either repaired by the non-homologous end-joining pathway (NHEJ), or by homology-directed repair (HDR). The HDR pathway allows for a precise site-specific knock-in and thus functional analysis of cancer-associated sequence variants. However, HDR activity is restricted to the late S and G2 phases of the cell cycle, while the NHEJ pathway dominates DNA repair throughout the cell cycle. Thus, the error-prone NHEJ pathway out-competes the HDR pathway, which reduces the likelihood for precise insertions, deletions or base substitutions at the DSB. Here, we devised a strategy to increase HDR by directly synchronizing the expression of Cas9 with cell cycle progression. Fusion of Cas9 to the N-terminal region of human Geminin converted this gene-editing protein into a substrate for the E3 ubiquitin ligase complex APC/Cdh1 resulting in a cell cycle tailored expression with low levels in G1, but high expression in S/G2/M. Importantly, Cas9-hGem(1/110) increased the relative rate of HDR by up to 87% at a single-copy reporter locus, and up to 42% at the endogenous MALAT1 locus compared to wild-type Cas9. In summary, our regulatory concept takes advantage of cell-autonomous pathways to enable fluctuation of Cas9 protein levels coupled to cell cycle dynamics. This resulted in higher site-specific integration events without sophisticated manipulation of cells by small molecules or additional gene targeting reagents. Future methodological developments may enable high-resolution expression of Cas9 and alternative genome engineering proteins like Cpf1 by combining cell cycle phase-specific transcriptional and post-translational regulatory elements. This might enhance HDR efficiencies even further, and will help to make sense of cancer genomic data. Citation Format: Tony Gutschner, Monika Hämmerle, Giannicola Genovese, Giulio F. Draetta, Lynda Chin. Cas9 protein engineering for cell cycle-specific genome editing to enhance homology directed repair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 71.

Probing the impact of chromatin conformation on genome editing tools.

Transcription activator-like effector nucleases (TALENs) and RNA-guided nucleases derived from clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 systems have become ubiquitous genome editing tools. Despite this, the impact that distinct high-order chromatin conformations have on these sequence-specific designer nucleases is, presently, ill-defined. The same applies to the relative performance of TALENs and CRISPR/Cas9 nucleases at isogenic target sequences subjected to different epigenetic modifications. Here, to address these gaps in our knowledge, we have implemented quantitative cellular systems based on genetic reporters in which the euchromatic and heterochromatic statuses of designer nuclease target sites are stringently controlled by small-molecule drug availability. By using these systems, we demonstrate that TALENs and CRISPR/Cas9 nucleases are both significantly affected by the high-order epigenetic context of their target sequences. In addition, this outcome could also be ascertained for S. pyogenes CRISPR/Cas9 complexes harbouring Cas9 variants whose DNA cleaving specificities are superior to that of the wild-type Cas9 protein. Thus, the herein investigated cellular models will serve as valuable functional readouts for screening and assessing the role of chromatin on designer nucleases based on different platforms or with different architectures or compositions.

The Asymmetric Interaction of Cas9 with Target DNA can Promote High Efficiency Homology‐Directed Genome Editing

Cas9 is an RNA‐guided endonuclease that has been adapted from its natural role in CRISPR‐mediated bacterial immunity to enable many applications demanding site‐specific DNA recognition. Wild type Cas9 has been rapidly embraced for genome engineering, either generating knockout cells and organisms via error‐prone non‐homologous end joining (NHEJ) or introducing precise edits via homology directed repair (HDR), while catalytically dead Cas9 (dCas9) fused to various effectors has been used for transcriptional regulation and genomic imaging. However, while Cas9's interaction with target DNA is central to its biological function and use as a genome targeting tool, the molecular nature of this complex is still poorly understood. We have found that the in vitro dissociation of wild type (wt) and dCas9 from double stranded DNA (dsDNA) are both remarkably slow, with a half‐life greater than six hours. However, this stable association is highly asymmetric, and wtCas9 releases only the PAM‐distal end of the non‐target DNA strand post‐cleavage while maintaining contact with the other three strands. dCas9 introduces a bubble into the target DNA that releases the non‐target strand and leaves it accessible for annealing by a complementary single stranded DNA (ssDNA). These activities are present during genome targeting in human cells and can be used to rationally design ssDNA templates that mediate highly efficient gene editing via annealing to the released strand. Optimized templates yield strand‐specific HDR rates over 50% and can furthermore mediate HDR from a single Cas9 nickase or dCas9. Our results highlight Cas9's unusually asymmetric interaction with target DNA, which could be functionally important for CRISPR spacer acquisition or viral interference. Our work also suggests that guiding the formation of specific DNA repair intermediates can dramatically increase the efficiency of genome editing. Because dCas9 is presumably incapable of introducing off‐target breaks in a genome, its use to stimulate HDR may be valuable for precision gene editing in certain therapeutic contexts.

Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9

The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets bearing a PAM (protospacer adjacent motif) and complementarity to the guide RNA. A recent study showed that, whereas wild-type Streptococcus pyogenes Cas9 (SpCas9) recognizes the 5'-NGG-3' PAM, the engineered VQR, EQR, and VRER SpCas9 variants recognize the 5'-NGA-3', 5'-NGAG-3', and 5'-NGCG-3' PAMs, respectively, thus expanding the targetable sequences in Cas9-mediated genome editing applications. Here, we present the high-resolution crystal structures of the three SpCas9 variants in complexes with a single-guide RNA and its altered PAM-containing, partially double-stranded DNA targets. A structural comparison of the three SpCas9 variants with wild-type SpCas9 revealed that the multiple mutations synergistically induce an unexpected displacement in the phosphodiester backbone of the PAM duplex, thereby allowing the SpCas9 variants to directly recognize the altered PAM nucleotides. Our findings explain the altered PAM specificities of the SpCas9 variants and establish a framework for further rational engineering of CRISPR-Cas9.

Post-translational Regulation of Cas9 during G1 Enhances Homology-Directed Repair.

CRISPR/Cas9 induces DNA double-strand breaks that are repaired by cell-autonomous repair pathways, namely, non-homologous end-joining (NHEJ), or homology-directed repair (HDR). While HDR is absent in G1, NHEJ is active throughout the cell cycle and, thus, is largely favored over HDR. We devised a strategy to increase HDR by directly synchronizing the expression of Cas9 with cell-cycle progression. Fusion of Cas9 to the N-terminal region of human Geminin converted this gene-editing protein into a substrate for the E3 ubiquitin ligase complex APC/Cdh1, resulting in a cell-cycle-tailored expression with low levels in G1 but high expression in S/G2/M. Importantly, Cas9-hGem(1/110) increased the rate of HDR by up to 87% compared to wild-type Cas9. Future developments may enable high-resolution expression of genome engineering proteins, which might increase HDR rates further, and may contribute to a better understanding of DNA repair pathways due to spatiotemporal control of DNA damage induction.

A CRISPR-Based Toolbox for Studying T Cell Signal Transduction.

CRISPR/Cas9 system is a powerful technology to perform genome editing in a variety of cell types. To facilitate the application of Cas9 in mapping T cell signaling pathways, we generated a toolbox for large-scale genetic screens in human Jurkat T cells. The toolbox has three different Jurkat cell lines expressing distinct Cas9 variants, including wild-type Cas9, dCas9-KRAB, and sunCas9. We demonstrated that the toolbox allows us to rapidly disrupt endogenous gene expression at the DNA level and to efficiently repress or activate gene expression at the transcriptional level. The toolbox, in combination with multiple currently existing genome-wide sgRNA libraries, will be useful to systematically investigate T cell signal transduction using both loss-of-function and gain-of-function genetic screens.

Cas9 Variants Expand the Target Repertoire in Caenorhabditis elegans.

The proliferation of CRISPR/Cas9-based methods in Caenorhabditis elegans has enabled efficient genome editing and precise genomic tethering of Cas9 fusion proteins. Experimental designs using CRISPR/Cas9 are currently limited by the need for a protospacer adjacent motif (PAM) in the target with the sequence NGG. Here we report the characterization of two modified Cas9 proteins in C. elegans that recognize NGA and NGCG PAMs. We found that each variant could stimulate homologous recombination with a donor template at multiple loci and that PAM specificity was comparable to that of wild-type Cas9. To directly compare effectiveness, we used CRISPR/Cas9 genome editing to generate a set of assay strains with a common single-guide RNA (sgRNA) target sequence, but that differ in the juxtaposed PAM (NGG, NGA, or NGCG). In this controlled setting, we determined that the NGA PAM Cas9 variant can be as effective as wild-type Cas9. We similarly edited a genomic target to study the influence of the base following the NGA PAM. Using four strains with four NGAN PAMs differing only at the fourth position and adjacent to the same sgRNA target, we observed that efficient homologous replacement was attainable with any base in the fourth position, with an NGAG PAM being the most effective. In addition to demonstrating the utility of two Cas9 mutants in C. elegans and providing reagents that permit CRISPR/Cas9 experiments with fewer restrictions on potential targets, we established a means to benchmark the efficiency of different Cas9::PAM combinations that avoids variations owing to differences in the sgRNA sequence.

A new age in functional genomics using CRISPR/Cas9 in arrayed library screening.

CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.

Abstract 1213: Endogenous p53 affinity tagging with CRISPR

Abstract Several antimetabolites used for cancer treatment, including hydroxyurea (HU) and pemetrexed (PTX), stabilize tumor suppressor protein p53, but fail to induce the transcription of p53 downstream target genes. To understand the underlying mechanism for the deficiency of stabilized p53, we are quantitatively characterizing p53 posttranslational modification and its interaction partners with mass spectrometry (MS). To facilitate the purification of endogenous p53, we established an HCT116 human colon carcinoma cell line, in which one allele of endogenous p53 was tagged with a classic Tandem Affinity Tag (TAP) at the C-terminus, using adenovirus associated virus (AAV) mediated homologous recombination (HR). The purification of p53 from this cell exhibited severe losses at each step, compromising analysis by MS. Because of this limited yield from TAP purification, we performed a systematic tagging of p53 with 3xFLAG, Strep-II, and HALO tags, and their combinations, at both N- and C-termini. The latest CRISPR (clustered regularly interspaced short palindromic repeats)/cas9 nuclease system provides major advantages for endogenous gene modification through HR. Efficient site-specific protein tagging (or mutation) requires a DNA break point in the immediate vicinity, limiting the choice of guide DNA (gDNA) positions possible. By extending the complementary sequence between CRISPR targeting RNA (crRNA) and trans-activating crRNA (tracrRNA) in the chimeric single guide RNA (sgRNA), we expanded the essential Protospacer Adjacent Motif (PAM) sequence of the S. pyogenes CRISPR II system from NGG to NAG with equal targeting efficiency. Cas9 nickase (both D10A, and H840A-N854A-N863A) generated more favorable HR rates compared to non-homologous end joining (NHEJ), while mitigating off-target effects, in comparison to wild type cas9. Utilizing our conditions, we achieved 2-8% (for different tags) endogenous p53 tagging in the background of 10-60% NHEJ from wild type cas9 and single sgRNA. With cas9 nickase and two sgRNA, we consistently achieved 0.2-2% specific p53 tagging in the background of 0.01-8% NHEJ, with higher efficiency for 5′ than 3′ single strand overhangs generated. We are currently generating single cell clones, selecting those tags most faithful to natural p53 expression levels, and testing p53 purification efficiencies with different tags. In conclusion, we optimized the CRISPR system, extensively characterized the individual steps in generating single cell clones for endogenous gene tagging, and will apply the optimal tag to purify p53 and its co-eluted binding partners for quantitative MS analysis. Citation Format: Chen Yang, Cortney L. Lawrence, Charles E. Lyons, Shirley M. Taylor, Richard G. Moran. Endogenous p53 affinity tagging with CRISPR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1213. doi:10.1158/1538-7445.AM2015-1213

Development of an intein-mediated split-Cas9 system for gene therapy.

Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split–Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted. We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes. An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A. Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR). In addition, we explored the possibility of delivering the repair template accommodated on the same dual-plasmid system, by transient transfection, showing an efficient HDR. Most importantly, we revealed for the first time that intein-mediated split–Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.

119. HIV-1 Proviral DNA Excision and Deactivation Using a CRISPR/Cas9 “Nickase” Targeted to the 5’ and 3’ LTR

Permanent integration of the HIV-1 provirus in the host genome ensures a persistent supply of latently infected HIV cells capable of reactivation. This latent reservoir is recalcitrant to antiretroviral therapy (ART) making lifelong treatment the only option for patients. Therapies aimed at targeting the latent virus offer a promising approach to a “functional cure” of HIV/AIDS. Several gene editing technologies, including ZFNs, TALENs, Tre-recombinases and RNA-guided CRISPR/Cas9 tools have been used to disrupt the HIV-1 genome and suppress viral gene expression and replication in latently-infected cells. While CRISPR/Cas9 represents a powerful and facile DNA editing technology, toxicities associated with the Cas9 nuclease activity remains a concern. Here we explore the use of a “nickase” Cas9 (nCas9), which has a mutated RuvC I catalytic domain, and requires the combination of two adjacent small guide RNAs (sgRNAs) for on-target editing and specificity. We initially identified 9 sgRNA sequences targeted to conserved sites within the HIV TAR element, found on both the 5’ and 3’ LTRs. Using integrated and episomal LTR-driven luciferase reporters, we identified functional sgRNA “nickase pairs” capable of inducing an 80% reduction in gene expression, as well as excision of the proviral DNA. Using HIV-1 latency models, plasmids expressing nCas9 and the sgRNA pairs were nucleofected into HIV-1 integrated cell lines J1.1, CHME5 and U1 and treated with the histone deacetylase inhibitor trichostatin A (TSA) to activate infection. All treated cells showed significant reduction (up to 80% for J1.1) in infectious viral output. Cleavage was further verified using ChIP assays to detect DNA damage response proteins recruited to the targeted locus in all cell lines. Lastly, using a custom algorithm for detecting off-target cleavage sites (https://cm.jefferson.edu/Off-Spotter/), we identified 8 sites for each sgRNA within the “nickase pair”. No off-target editing was observed at these loci following high throughput NGS analysis. In conclusion, we showed that CRISPR/Cas technology represents a powerful tool at eradicating and/or deactivating the integrated HIV-1 provirus and that using the Cas9 nickase is just as effective as wild-type Cas9 but with the added advantage of providing a significant additional layer of safety for future in vivo applications.

Small molecule-triggered Cas9 protein with improved genome-editing specificity.

Directly modulating the activity of genome-editing proteins has the potential to increase their specificity by reducing activity following target locus modification. We developed Cas9 nucleases that are activated by the presence of a cell-permeable small molecule by inserting an evolved 4-hydroxytamoxifen (4-HT)-responsive intein at specific positions in Cas9. In human cells, conditionally active Cas9s modify target genomic sites with up to 25-fold higher specificity than wild-type Cas9.

Detailed phenotypic and molecular analyses of genetically modified mice generated by CRISPR-Cas9-mediated editing.

The bacterial CRISPR-Cas9 system has been adapted for use as a genome editing tool. While several recent reports have indicated that successful genome editing of mice can be achieved, detailed phenotypic and molecular analyses of the mutant animals are limited. Following pronuclear micro-injection of fertilized eggs with either wild-type Cas9 or the nickase mutant (D10A) and single or paired guide RNA (sgRNA) for targeting of the tyrosinase (Tyr) gene, we assessed genome editing in mice using rapid phenotypic readouts (eye and coat color). Mutant mice with insertions or deletions (indels) in Tyr were efficiently generated without detectable off-target cleavage events. Gene correction of a single nucleotide by homologous recombination (HR) could only occur when the sgRNA recognition sites in the donor DNA were modified. Gene repair did not occur if the donor DNA was not modified because Cas9 catalytic activity was completely inhibited. Our results indicate that allelic mosaicism can occur following -Cas9-mediated editing in mice and appears to correlate with sgRNA cleavage efficiency at the single-cell stage. We also show that larger than expected deletions may be overlooked based on the screening strategy employed. An unbiased analysis of all the deleted nucleotides in our experiments revealed that the highest frequencies of nucleotide deletions were clustered around the predicted Cas9 cleavage sites, with slightly broader distributions than expected. Finally, additional analysis of founder mice and their offspring indicate that their general health, fertility, and the transmission of genetic changes were not compromised. These results provide the foundation to interpret and predict the diverse outcomes following CRISPR-Cas9-mediated genome editing experiments in mice.

453 Plasma metabolomic signature of novel signal transduction inhibitors from preclinical identification to clinical validation

function aMOI and a NF1 loss of function aMOI, while SW694 has a NF1 loss of function aMOI, but these mutations do not correlate with sensitivity to trametinib. Interestingly, HT1080 has a heterozygous mutation in NRAS (Q61K) and mutations in NRAS have been shown to correlate with sensitivity to trametinib in cell lines with BRAF wild type background. To validate whether NRAS Q61K determines sensitivity to trametinib in HT1080, we used the CRISPR/Cas9 system to repair NRAS Q61K by homologous recombination. The system utilizes the type II prokaryotic CRISPR/Cas9 adaptive immune system and targets the Cas9 nuclease by a 20 nucleotides guide sequence cloned upstream of a 50-NGG “protospacer adjacent motif” (PAM). The induced site-specific strand breaks are repaired either by nonhomologous end-joining (NHEJ) to yield indels or by homologous recombination (HR) if homologous donor templates are available, thereby enhancing the efficiency of HR-based gene targeting. We designed several different small guide RNA sequences and we verified that all sequences were able to introduce indels in the NRAS gene using a control cell line. We therefore used either wild type Cas9 or Cas9D10A (nickase) with donor DNAs to perform NRAS gene repair. Clones are actively being screened for NRAS repair. Results for these experiments will be presented in the poster. The combination of drug sensitivity data, together with the mutation sequencing and precise gene editing using the CRISPR/Cas9 system may lead to precise targeting of genomic errors and to the identification of more effective therapeutic strategies for sarcoma. Funded by NCI Contract No. HHSN261200800001E. This research was supported [in part] by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute.