Diana F. Colgan

Unexpected mutations after CRISPR-Cas9 editing in vivo

To the Editor: CRISPR–Cas9 editing shows promise for correcting disease-causing mutations. For example, in a recent study we used CRISPR-Cas9 for sight restoration in blind rd1 mice by correcting a mutation in the Pde6b gene1. However, concerns persist regarding secondary mutations in regions not targeted by the single guide RNA (sgRNA)2. Algorithms generate likely off-target sites for a given gRNA, but these algorithms may miss mutations. Whole-genome sequencing (WGS) has been used to assess the presence of small insertions and deletions (indels)3 but not to probe for single-nucleotide variants (SNVs) in a whole organism. We performed WGS on a CRISPR–Cas9-edited mouse to identify all off-target mutations and found an unexpectedly high number of SNVs compared with the widely accepted assumption that CRISPR causes mostly indels at regions homologous to the sgRNA. We tested four sgRNAs in cells then chose the sgRNA with the highest activity for in vivo targeting. DNA was isolated from two CRISPR-repaired mice (F03 and F05) and one uncorrected control1. CRISPR–Cas9-treated mice were sequenced at an average depth of 50×, and the control was sequenced at 30×. Variant calls were confirmed by at least 23× sequencing coverage (Supplementary Tables 1 and 2). Multiple variant-calling software pipelines identified indels and SNVs (Fig. 1 and Supplementary Methods). In the CRISPR-treated mice, targeted alleles were repaired1. Off-target mutations were identified as those present in the CRISPR-treated animals but absent in the uncorrected control. All pipelines showed that F03 harbored 164 indels and 1,736 SNVs (63 and 885 of these, respectively, associated with known genes). F05 harbored 128 indels and 1,696 SNVs (51 and 865 of these, respectively, associated with known genes) (Fig. 1). The same 117 indels and 1,397 SNVs were detected in both of the CRISPR-treated mice, which indicated nonrandom targeting. SNVs appeared to slightly favor transitions over transversions (Supplementary Fig. 1). The mutation rate detected in CRISPRtreated mice was substantially higher than that generated by spontaneous germline mutations (3 to 4 indels and 90 to 100 SNVs, de novo, per generation)4,5. As additional controls, each of the variants was compared with the FVB/NJ genome in the mouse dbSNP database (v138), and each of the SNVs was also compared with all 36 strains in the Mouse Genome Project (v3). None of the CRISPR-generated offtarget mutations were found in any of these strains, which further confirmed that these WGS-identified SNVs were the result of CRISPR–Cas9 off targeting. All pipelines identified 6 and 3 indels and 60 and 51 SNVs in F03 and F05 mice, respectively, in exonic regions only (Fig. 1); 5 indels and 24 SNVs caused nonsynonymous mutations in protein-coding sequences (Supplementary Tables 3 and 4). Of these, all five indels and one SNV (introducing a premature stop codon) were expected to be deleterious. Several mutated protein-coding genes were associated with a human and/ or mouse phenotype (Supplementary Tables 3 and 4). Of the 29 coding-sequence variants, 7 variants were mutated identically in both mice. 24 CRISPR-associated variants were selected, and all were confirmed by Sanger sequencing (Supplementary Fig. 2 and Supplementary Methods). Among the top-fifty sequences predicted for off targeting, none were mutated. Additionally, there was poor sequence homology between the sgRNA and sequences near the actual off-target coding and noncoding variants (Supplementary Fig. 3). Our results suggest current in silico modeling cannot predict bona fide off-target sites. Together, these results indicate that at least certain sgRNAs may target loci independently of their target in vivo. The unpredictable generation of these variants is of concern. The impact of the numerous mutations occurring in noncoding RNAs or other regulatory intragenic regions could be detrimental to key cellular processes (Supplementary Fig. 4 and Supplementary Table 5)6. Although our CRISPR-treated mice did not display obvious extraocular phenotypes, it is possible the mice may reveal phenotypes in time, when they are challenged or bred to homozygosity. The present study demonstrates WGS analysis of both indels and SNVs as the most thorough method for identifying off-target mutations and shows a significantly higher number of potentially deleterious CRISPR–Cas9-induced mutations than have been previously reported3. It is not clear whether improved sgRNA

Precision Medicine: Personalized Proteomics for the Diagnosis and Treatment of Idiopathic Inflammatory Disease

IMPORTANCE To better characterize posterior uveitis, vitreous samples from 15 patients were subjected to antibody arrays, and the expression levels of 200 human cytokines were evaluated. Expression was analyzed by 1-way analysis of variance (significance at P < .01), unsupervised cluster algorithm, and pathway analysis. OBSERVATIONS Unbiased clustering of patients, based on their cytokine expression profile, suggested that particular protein networks and molecular pathways are altered in various forms of uveitis. Expression of interleukin 23 (IL-23), IL-1 receptor I (IL-1RI), IL-17R, tissue inhibitors of metalloproteinase 1 and 2 (TIMP-1 and TIMP-2), insulinlike growth factor-binding protein 2 (IGFBP-2), nerve growth factor (b-NGF), platelet-derived growth factor receptor β polypeptide (PDGFRb), bone morphogenic protein 4 (BMP-4), and stem cell factor (SCF) constituted a common cytokine signature in the vitreous of patients with uveitis. In 1 patient with progressive, idiopathic visual loss, this last-line analysis implicated retinal autoimmunity, a diagnosis that was validated when her serum sample was found to contain antibodies to S-arrestin, a retinal protein and potent cause of autoimmune retinal degeneration. CONCLUSIONS AND RELEVANCE The analysis identifies a common cytokine signature for posterior uveitis and guides the diagnosis of a patient with idiopathic uveitis. Personalized treatment reversed the visual loss, illustrating how proteomic tools may individualize therapy.

Calpain-5 Expression in the Retina Localizes to Photoreceptor Synapses

Purpose We characterize calpain-5 (CAPN5) expression in retinal and neuronal subcellular compartments. Methods CAPN5 gene variants were classified using the exome variant server, and RNA-sequencing was used to compare expression of CAPN5 mRNA in the mouse and human retina and in retinoblastoma cells. Expression of CAPN5 protein was ascertained in humans and mice in silico, in mouse retina by immunohistochemistry, and in neuronal cancer cell lines and fractionated central nervous system tissue extracts by Western analysis with eight antibodies targeting different CAPN5 regions. Results Most CAPN5 genetic variation occurs outside its protease core; and searches of cancer and epilepsy/autism genetic databases found no variants similar to hyperactivating retinal disease alleles. The mouse retina expressed one transcript for CAPN5 plus those of nine other calpains, similar to the human retina. In Y79 retinoblastoma cells, the level of CAPN5 transcript was very low. Immunohistochemistry detected CAPN5 expression in the inner and outer nuclear layers and at synapses in the outer plexiform layer. Western analysis of fractionated retinal extracts confirmed CAPN5 synapse localization. Western blots of fractionated brain neuronal extracts revealed distinct subcellular patterns and the potential presence of autoproteolytic CAPN5 domains. Conclusions CAPN5 is moderately expressed in the retina and, despite higher expression in other tissues, hyperactive disease mutants of CAPN5 only manifest as eye disease. At the cellular level, CAPN5 is expressed in several different functional compartments. CAPN5 localization at the photoreceptor synapse and with mitochondria explains the neural circuitry phenotype in human CAPN5 disease alleles.

Corrigendum and follow-up: Whole genome sequencing of multiple CRISPR-edited mouse lines suggests no excess mutations.

Our previous publication suggested CRISPR-Cas9 editing at the zygotic stage might unexpectedly introduce a multitude of subtle but unintended mutations, an interpretation that not surprisingly raised numerous questions. The key issue is that since parental lines were not available, might the reported variants have been inherited? To expand upon the limited available whole genome data on whether CRISPR-edited mice show more genetic variation, whole-genome sequencing was performed on two other mouse lines that had undergone a CRISPR-editing procedure. Again, parents were not available for either the Capn5 nor Fblim1 CRISPR-edited mouse lines, so strain controls were examined. Additionally, we also include verification of variants detected in the initial mouse line. Taken together, these whole-genome-sequencing-level results support the idea that in specific cases, CRISPR-Cas9 editing can precisely edit the genome at the organismal level and may not introduce numerous, unintended, off-target mutations.Here we provide additional confirmatory data and clarifying discussion, including sequencing data showing extensive heterozygous mutations throughout the genome in the CRISPR treated mice, which are all progeny of inbred mice purchased from a commercial vendor (JAX). The heterozygosity in these cases cannot be parentally inherited. The summary statements in our Correspondence reflect observations of a secondary outcome following successful achievement of the primary outcome using CRISPR to treat blindness in Pde6b/rd1 mice. As the scientific community considers the role of WGS in off-target analysis, future in vivo studies are needed where the design and primary outcome focuses on CRISPR off-targeting. We agree that a range of WGS controls are needed that include parents, different gRNAs, different versions of Cas9, and different in vivo protocols. We look forward to the publication of such studies. Combined, these results will be essential to fully understand off-targeting and can be used to create better algorithms for off-target prediction. Overall, we are optimistic that some form of CRISPR therapy will be successfully engineered to treat blindness.Here we provide additional confirmatory data and clarifying discussion, including information about the relatedness of the control and CRISPR-treated mice, as well as sequencing data showing extensive regions with heterozygosity and in some cases more than 2 alleles in CRISPR-treated mice throughout the genome. The control FVB mouse and parents of the CRISPR-treated mice were purchased directly through the JAX genetic stability program and were within one generation of one another. Furthermore, the heterozygous mutations were mostly within 7-10bp adjacent to NGG or NGA nucleotide sequences, the preferred Protospacer Adjacent Motif (PAM) for the SpCas9. The multiple alleles in these cases cannot simply be explained by parental inheritance. The summary statements in our Correspondence reflect observations of a secondary outcome following successful achievement of the primary outcome using CRISPR to treat blindness in Pde6b/rd1 mice. As the scientific community considers the role of WGS in off-target analysis, future in vivo studies are needed where the design and primary outcome focuses on CRISPR off-targeting. We agree that a range of WGS controls are needed that include parents, different gRNAs, different versions of Cas9, and different in vivo protocols. We look forward to the publication of such studies. Combined, these results will be essential to fully understand off-targeting and can be used to create better algorithms for off-target prediction. Overall, we are optimistic that some form of CRISPR therapy will be successfully engineered to treat blindness.

Therapeutic drug repositioning using personalized proteomics of liquid biopsies

BACKGROUND In patients with limited response to conventional therapeutics, repositioning of already approved drugs can bring new, more effective options. Current drug repositioning methods, however, frequently rely on retrospective computational analyses and genetic testing - time consuming methods that delay application of repositioned drugs. Here, we show how proteomic analysis of liquid biopsies successfully guided treatment of neovascular inflammatory vitreoretinopathy (NIV), an inherited autoinflammatory disease with otherwise poor clinical outcomes. METHODS Vitreous biopsies from NIV patients were profiled by an antibody array for expression of 200 cytokine-signaling proteins. Non-NIV controls were compared with NIV samples from various stages of disease progression. Patterns were identified by 1-way ANOVA, hierarchical clustering, and pathway analysis. Subjects treated with repositioned therapies were followed longitudinally. RESULTS Proteomic profiles revealed molecular pathways in NIV pathologies and implicated superior and inferior targets for therapy. Anti-VEGF injections resolved vitreous hemorrhages without the need for vitrectomy surgery. Methotrexate injections reversed inflammatory cell reactions without the side effects of corticosteroids. Anti-IL-6 therapy prevented recurrent fibrosis and retinal detachment where all prior antiinflammatory interventions had failed. The cytokine array also showed that TNF-α levels were normal and that corticosteroid-sensitive pathways were absent in fibrotic NIV, helping explain prior failure of these conventional therapeutic approaches. CONCLUSIONS Personalized proteomics can uncover highly personalized therapies for autoinflammatory disease that can be timed with specific pathologic activities. This precision medicine strategy can also help prevent delivery of ineffective drugs. Importantly, proteomic profiling of liquid biopsies offers an endpoint analysis that can directly guide treatment using available drugs.

Response to Editas and Intellia: Unexpected mutations after CRISPR-Cas9 editing in vivo

Our previous publication suggested CRISPR-Cas9 editing at the zygotic stage might unexpectedly introduce a multitude of subtle but unintended mutations, an interpretation that not surprisingly raised numerous questions. The key issue is that since parental lines were not available, might the reported variants have been inherited? To expand upon the limited available whole genome data on whether CRISPR-edited mice show more genetic variation, whole-genome sequencing was performed on two other mouse lines that had undergone a CRISPR-editing procedure. Again, parents were not available for either the Capn5 nor Fblim1 CRISPR-edited mouse lines, so strain controls were examined. Additionally, we also include verification of variants detected in the initial mouse line. Taken together, these whole-genome-sequencing-level results support the idea that in specific cases, CRISPR-Cas9 editing can precisely edit the genome at the organismal level and may not introduce numerous, unintended, off-target mutations.Here we provide additional confirmatory data and clarifying discussion, including sequencing data showing extensive heterozygous mutations throughout the genome in the CRISPR treated mice, which are all progeny of inbred mice purchased from a commercial vendor (JAX). The heterozygosity in these cases cannot be parentally inherited. The summary statements in our Correspondence reflect observations of a secondary outcome following successful achievement of the primary outcome using CRISPR to treat blindness in Pde6b/rd1 mice. As the scientific community considers the role of WGS in off-target analysis, future in vivo studies are needed where the design and primary outcome focuses on CRISPR off-targeting. We agree that a range of WGS controls are needed that include parents, different gRNAs, different versions of Cas9, and different in vivo protocols. We look forward to the publication of such studies. Combined, these results will be essential to fully understand off-targeting and can be used to create better algorithms for off-target prediction. Overall, we are optimistic that some form of CRISPR therapy will be successfully engineered to treat blindness.Here we provide additional confirmatory data and clarifying discussion, including information about the relatedness of the control and CRISPR-treated mice, as well as sequencing data showing extensive regions with heterozygosity and in some cases more than 2 alleles in CRISPR-treated mice throughout the genome. The control FVB mouse and parents of the CRISPR-treated mice were purchased directly through the JAX genetic stability program and were within one generation of one another. Furthermore, the heterozygous mutations were mostly within 7-10bp adjacent to NGG or NGA nucleotide sequences, the preferred Protospacer Adjacent Motif (PAM) for the SpCas9. The multiple alleles in these cases cannot simply be explained by parental inheritance. The summary statements in our Correspondence reflect observations of a secondary outcome following successful achievement of the primary outcome using CRISPR to treat blindness in Pde6b/rd1 mice. As the scientific community considers the role of WGS in off-target analysis, future in vivo studies are needed where the design and primary outcome focuses on CRISPR off-targeting. We agree that a range of WGS controls are needed that include parents, different gRNAs, different versions of Cas9, and different in vivo protocols. We look forward to the publication of such studies. Combined, these results will be essential to fully understand off-targeting and can be used to create better algorithms for off-target prediction. Overall, we are optimistic that some form of CRISPR therapy will be successfully engineered to treat blindness.

Catenin delta-1 (CTNND1) phosphorylation controls the mesenchymal to epithelial transition in astrocytic tumors.

Inactivating mutations of the TSC1/TSC2 complex (TSC1/2) cause tuberous sclerosis (TSC), a hereditary syndrome with neurological symptoms and benign hamartoma tumours in the brain. Since TSC effectors are largely unknown in the human brain, TSC patient cortical tubers were used to uncover hyperphosphorylation unique to TSC primary astrocytes, the cell type affected in the brain. We found abnormal hyperphosphorylation of catenin delta-1 S268, which was reversible by mTOR-specific inhibitors. In contrast, in three metastatic astrocytoma cell lines, S268 was under phosphorylated, suggesting S268 phosphorylation controls metastasis. TSC astrocytes appeared epithelial (i.e. tightly adherent, less motile, and epithelial (E)-cadherin positive), whereas wild-type astrocytes were mesenchymal (i.e. E-cadherin negative and highly motile). Despite their epithelial phenotype, TSC astrocytes outgrew contact inhibition, and monolayers sporadically generated tuberous foci, a phenotype blocked by the mTOR inhibitor, Torin1. Also, mTOR-regulated phosphokinase C epsilon (PKCe) activity induced phosphorylation of catenin delta-1 S268, which in turn mediated cell-cell adhesion in astrocytes. The mTOR-dependent, epithelial phenotype of TSC astrocytes suggests TSC1/2 and mTOR tune the phosphorylation level of catenin delta-1 by controlling PKCe activity, thereby regulating the mesenchymal-epithelial-transition (MET). Thus, some forms of TSC could be treated with PKCe inhibitors, while metastasis of astrocytomas might be blocked by PKCe stimulators.

Response to Editas: Unexpected mutations after CRISPR-Cas9 editing in vivo

Our previous publication suggested CRISPR-Cas9 editing at the zygotic stage might unexpectedly introduce a multitude of subtle but unintended mutations, an interpretation that not surprisingly raised numerous questions. The key issue is that since parental lines were not available, might the reported variants have been inherited? To expand upon the limited available whole genome data on whether CRISPR-edited mice show more genetic variation, whole-genome sequencing was performed on two other mouse lines that had undergone a CRISPR-editing procedure. Again, parents were not available for either the Capn5 nor Fblim1 CRISPR-edited mouse lines, so strain controls were examined. Additionally, we also include verification of variants detected in the initial mouse line. Taken together, these whole-genome-sequencing-level results support the idea that in specific cases, CRISPR-Cas9 editing can precisely edit the genome at the organismal level and may not introduce numerous, unintended, off-target mutations.Here we provide additional confirmatory data and clarifying discussion, including sequencing data showing extensive heterozygous mutations throughout the genome in the CRISPR treated mice, which are all progeny of inbred mice purchased from a commercial vendor (JAX). The heterozygosity in these cases cannot be parentally inherited. The summary statements in our Correspondence reflect observations of a secondary outcome following successful achievement of the primary outcome using CRISPR to treat blindness in Pde6b/rd1 mice. As the scientific community considers the role of WGS in off-target analysis, future in vivo studies are needed where the design and primary outcome focuses on CRISPR off-targeting. We agree that a range of WGS controls are needed that include parents, different gRNAs, different versions of Cas9, and different in vivo protocols. We look forward to the publication of such studies. Combined, these results will be essential to fully understand off-targeting and can be used to create better algorithms for off-target prediction. Overall, we are optimistic that some form of CRISPR therapy will be successfully engineered to treat blindness.Here we provide additional confirmatory data and clarifying discussion, including information about the relatedness of the control and CRISPR-treated mice, as well as sequencing data showing extensive regions with heterozygosity and in some cases more than 2 alleles in CRISPR-treated mice throughout the genome. The control FVB mouse and parents of the CRISPR-treated mice were purchased directly through the JAX genetic stability program and were within one generation of one another. Furthermore, the heterozygous mutations were mostly within 7-10bp adjacent to NGG or NGA nucleotide sequences, the preferred Protospacer Adjacent Motif (PAM) for the SpCas9. The multiple alleles in these cases cannot simply be explained by parental inheritance. The summary statements in our Correspondence reflect observations of a secondary outcome following successful achievement of the primary outcome using CRISPR to treat blindness in Pde6b/rd1 mice. As the scientific community considers the role of WGS in off-target analysis, future in vivo studies are needed where the design and primary outcome focuses on CRISPR off-targeting. We agree that a range of WGS controls are needed that include parents, different gRNAs, different versions of Cas9, and different in vivo protocols. We look forward to the publication of such studies. Combined, these results will be essential to fully understand off-targeting and can be used to create better algorithms for off-target prediction. Overall, we are optimistic that some form of CRISPR therapy will be successfully engineered to treat blindness.

Deeper sequencing at unexpected CRISPR/Cas9 off-target sites in vivo : A response to Editas, Intellia, Beacon, ToolGen and others

Our previous publication suggested CRISPR-Cas9 editing at the zygotic stage might unexpectedly introduce a multitude of subtle but unintended mutations, an interpretation that not surprisingly raised numerous questions. The key issue is that since parental lines were not available, might the reported variants have been inherited? To expand upon the limited available whole genome data on whether CRISPR-edited mice show more genetic variation, whole-genome sequencing was performed on two other mouse lines that had undergone a CRISPR-editing procedure. Again, parents were not available for either the Capn5 nor Fblim1 CRISPR-edited mouse lines, so strain controls were examined. Additionally, we also include verification of variants detected in the initial mouse line. Taken together, these whole-genome-sequencing-level results support the idea that in specific cases, CRISPR-Cas9 editing can precisely edit the genome at the organismal level and may not introduce numerous, unintended, off-target mutations.Here we provide additional confirmatory data and clarifying discussion, including sequencing data showing extensive heterozygous mutations throughout the genome in the CRISPR treated mice, which are all progeny of inbred mice purchased from a commercial vendor (JAX). The heterozygosity in these cases cannot be parentally inherited. The summary statements in our Correspondence reflect observations of a secondary outcome following successful achievement of the primary outcome using CRISPR to treat blindness in Pde6b/rd1 mice. As the scientific community considers the role of WGS in off-target analysis, future in vivo studies are needed where the design and primary outcome focuses on CRISPR off-targeting. We agree that a range of WGS controls are needed that include parents, different gRNAs, different versions of Cas9, and different in vivo protocols. We look forward to the publication of such studies. Combined, these results will be essential to fully understand off-targeting and can be used to create better algorithms for off-target prediction. Overall, we are optimistic that some form of CRISPR therapy will be successfully engineered to treat blindness.Here we provide additional confirmatory data and clarifying discussion, including information about the relatedness of the control and CRISPR-treated mice, as well as sequencing data showing extensive regions with heterozygosity and in some cases more than 2 alleles in CRISPR-treated mice throughout the genome. The control FVB mouse and parents of the CRISPR-treated mice were purchased directly through the JAX genetic stability program and were within one generation of one another. Furthermore, the heterozygous mutations were mostly within 7-10bp adjacent to NGG or NGA nucleotide sequences, the preferred Protospacer Adjacent Motif (PAM) for the SpCas9. The multiple alleles in these cases cannot simply be explained by parental inheritance. The summary statements in our Correspondence reflect observations of a secondary outcome following successful achievement of the primary outcome using CRISPR to treat blindness in Pde6b/rd1 mice. As the scientific community considers the role of WGS in off-target analysis, future in vivo studies are needed where the design and primary outcome focuses on CRISPR off-targeting. We agree that a range of WGS controls are needed that include parents, different gRNAs, different versions of Cas9, and different in vivo protocols. We look forward to the publication of such studies. Combined, these results will be essential to fully understand off-targeting and can be used to create better algorithms for off-target prediction. Overall, we are optimistic that some form of CRISPR therapy will be successfully engineered to treat blindness.

Early B Cell Progenitors Deficient for GON4L Fail To Differentiate Due to a Block in Mitotic Cell Division

B cell development in Justy mutant mice is blocked due to a precursor mRNA splicing defect that depletes the protein GON4-like (GON4L) in B cell progenitors. Genetic and biochemical studies have suggested that GON4L is a transcriptional regulator that coordinates cell division with differentiation, but its role in B cell development is unknown. To understand the function of GON4L, we characterized B cell differentiation, cell cycle control, and mitotic gene expression in GON4L-deficient B cell progenitors from Justy mice. We found that these cells established key aspects of the transcription factor network that guides B cell development and proliferation and rearranged the IgH gene locus. However, despite intact IL-7 signaling, GON4L-deficient pro-B cell stage precursors failed to undergo a characteristic IL-7–dependent proliferative burst. These cells also failed to upregulate genes required for mitotic division, including those encoding the G1/S cyclin D3 and E2F transcription factors and their targets. Additionally, GON4L-deficient B cell progenitors displayed defects in DNA synthesis and passage through the G1/S transition, contained fragmented DNA, and underwent apoptosis. These phenotypes were not suppressed by transgenic expression of prosurvival factors. However, transgenic expression of cyclin D3 or other regulators of the G1/S transition restored pro-B cell development from Justy progenitor cells, suggesting that GON4L acts at the beginning of the cell cycle. Together, our findings indicate that GON4L is essential for cell cycle progression and division during the early stages of B cell development.