Genome editing of HBG1 and HBG2 to induce fetal hemoglobin

Induction of fetal hemoglobin (HbF) via clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of DNA regulatory elements that repress gamma-globin gene (HBG1 and HBG2) expression is a promising therapeutic strategy for sickle cell disease (SCD) and beta-thalassemia, although the optimal technical approaches and limiting toxicities are not yet fully defined. We disrupted an HBG1/HBG2 gene promoter motif that is bound by the transcriptional repressor BCL11A. Electroporation of Cas9 single guide RNA ribonucleoprotein complex into normal and SCD donor CD34+ hematopoietic stem and progenitor cells resulted in high frequencies of on-target mutations and the induction of HbF to potentially therapeutic levels in erythroid progeny generated in vitro and in vivo after transplantation of hematopoietic stem and progenitor cells into nonobese diabetic/severe combined immunodeficiency/Il2rgamma-/-/KitW41/W41 immunodeficient mice. On-target editing did not impair CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages at 16 to 17 weeks after xenotransplantation. No off-target mutations were detected by targeted sequencing of candidate sites identified by circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), an in vitro genome-scale method for detecting Cas9 activity. Engineered Cas9 containing 3 nuclear localization sequences edited human hematopoietic stem and progenitor cells more efficiently and consistently than conventional Cas9 with 2 nuclear localization sequences. Our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a mechanism-based approach to induce HbF for treating hemoglobinopathies

Broad Specificity Profiling of TALENs Results in Engineered Nucleases With Improved DNA Cleavage Specificity

Although transcription activator-like effector nucleases (TALENs) can be designed to cleave chosen DNA sequences, TALENs have been shown to have activity against related off-target sequences. To better understand TALEN specificity and engineer TALENs with improved specificity, we profiled 30 unique TALENs with varying target sites, array length, and domain sequences for their ability to cleave any of 1012 potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells. The results collectively suggest that (i) TALE repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches, yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity in cells. Based on these findings, we engineered a TALEN variant, Q3, that exhibits equal on-target cleavage activity but 10-fold lower average off-target activity in human cells. Our results demonstrate that identifying and mutating residues that contribute to non-specific DNA-binding can yield genome editing reagents with improved DNA specificities

Altering, Improving, And Defining The Specificities Of Crispr-Cas Nucleases

CRISPR-Cas9 nucleases have been widely adopted for genome editing applications to knockout genes or to introduce desired changes. While these nucleases have shown immense promise, two notable limitations of the wild-type form of the broadly used Streptococcus pyogenes Cas9 (SpCas9) are the restriction of targeting range to sites that contain an NGG protospacer adjacent motif (PAM), and the undesirable ability of the enzyme to cleave off-target sites that resemble the on-target site. Scarcity of PAM motifs can limit implementations that require precise targeting, whereas off-target effects can confound research applications and are important considerations for therapeutics. To improve the targeting range of SpCas9 and an orthogonal Cas9 from Staphylococcus aureus (called SaCas9), we optimized a heterologous genetic selection system that enabled us to perform directed evolution of PAM specificity. With SpCas9, we evolved two separate variants that can target NGA and NGCG PAMs1, and with SaCas9 relaxed the PAM from NNGRRT to NNNRRT2, increasing the targetability of these enzyme 2- to 4-fold. The genome-wide specificity profiles of SpCas9 and SaCas9 variants, determine by GUIDE-seq3, indicate that they are at least as, if not more, specific than the wild-type enzyme1,2. Together, these results demonstrate that the inherent PAM specificity of multiple different Cas9 orthologues can be purposefully modified to improve the accuracy of targeting. Existing strategies for improving the genome-wide specificity of SpCas9 have thus far proven to be incompletely effective and/or have other limitations that constrain their use. To address the off-target potential of SpCas9, we engineered a high-fidelity variant of SpCas9 (called SpCas9-HF1), that contains alterations designed to reduce non-specific contacts to the target strand DNA backbone. In comparison to wild-type SpCas9, SpCas9-HF1 rendered all or nearly all off-target events imperceptible by GUIDE-seq and targeted deep-sequencing methods with standard non-repetitive target sites in human cells4. Even for atypical, repetitive target sites, the vast majority of off-targets induced by SpCas9-HF1 and optimized derivatives were not detected4. With its exceptional precision, SpCas9-HF1 provides an important and easily employed alternative to wild-type SpCas9 that can eliminate off-target effects when using CRISPR-Cas9 for research and therapeutic applications. Finally, on-target activity and genome-wide specificity are two important properties of engineered nucleases that should be characterized prior to adoption of such technologies for research or therapeutic applications. CRISPR-Cas Cpf1 nucleases have recently been described as an alternative genome-editing platform5, yet their activities and genome-wide specificities remain largely undefined. Based on assessment of on-target activity across more than 40 target sites, we demonstrate that two Cpf1 orthologues function robustly in human cells with efficiencies comparable to those of the widely used Streptococcus pyogenes Cas9. We also demonstrate that four to six bases at the 3’ end of the short CRISPR RNA (crRNA) used to program Cpf1 are insensitive to single base mismatches, but that many of the other bases within the crRNA targeting region are highly sensitive to single or double substitutions6. Consistent with these results, GUIDE-seq performed in multiple cell types and targeted deep sequencing analyses of two Cpf1 nucleases revealed no detectable off-target cleavage for over half of 20 different crRNAs we examined. Our results suggest that the two Cpf1 nucleases we characterized generally possess robust on-target activity and high specificities in human cells, findings that should encourage broader use of these genome editing enzymes. 1. Kleinstiver, BP, et al. (2015) Nature, 523(7561):481-5 2. Kleinstiver, BP, et al. (2015) Nature Biotechnology, 33(12):1293-98 3. Tsai, SQ et al. (2015) Nature Biotechnology, 33(2):187-97 4. Kleinstiver, BP and Pattanayak, V, et al. (2016), Nature, 529(7587):490-5 5. Zetsche, B, et al. (2015) Cell, 163(3):759-71 6. Kleinstiver, BP and Tsai, SQ, et al. (2016), Nature Biotechnology, 34(8):869-7

Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing

Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI Nucleases (RFNs) that recognize extended sequences and can edit endogenous genes with high efficiencies in human cells. The cleavage activity of an RFN depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation and therefore show improved specificities relative to wild-type Cas9 monomers. Importantly, direct comparisons show that RFNs guided by a single gRNA generally induce lower levels of unwanted mutations than matched monomeric Cas9 nickases. In addition, we describe a simple method for expressing multiple gRNAs bearing any 5′ end nucleotide, which gives dimeric RFNs a broad targeting range. RFNs combine the ease of RNA-based targeting with the specificity enhancement inherent to dimerization and are likely to be useful in applications that require highly precise genome editing

Efficient In Vivo Genome Editing Using RNA-Guided Nucleases

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have evolved in bacteria and archaea as a defense mechanism to silence foreign nucleic acids of viruses and plasmids. Recent work has shown that bacterial type II CRISPR systems can be adapted to create guide RNAs (gRNAs) capable of directing site-specific DNA cleavage by the Cas9 nuclease in vitro. Here we show that this system can function in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies comparable to those obtained using ZFNs and TALENs for the same genes. RNA-guided nucleases robustly enabled genome editing at 9 of 11 different sites tested, including two for which TALENs previously failed to induce alterations. These results demonstrate that programmable CRISPR/Cas systems provide a simple, rapid, and highly scalable method for altering genes in vivo, opening the door to using RNA-guided nucleases for genome editing in a wide range of organisms

Efficient genome editing in zebrafish using a CRISPR-Cas system

In bacteria, foreign nucleic acids are silenced by clustered, regularly interspaced, short palindromic repeats (CRISPR)--CRISPR-associated (Cas) systems. Bacterial type II CRISPR systems have been adapted to create guide RNAs that direct site-specific DNA cleavage by the Cas9 endonuclease in cultured cells. Here we show that the CRISPR-Cas system functions in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies similar to those obtained using zinc finger nucleases and transcription activator-like effector nucleases

Virus-Free CRISPR CAR T cells induce solid tumor regression

Chimeric antigen receptor (CAR) T cell therapy has shown promising efficacy in treating hematologic malignancies and has led to the FDA-approval of three CAR T cell products. However, there has been little success in treating solid tumors, as clinical trials to date have yielded little to no responses and no improvement in survival. Current methods of CAR T cell production typically involve the use of viral vectors which can give rise to complications such as insertional mutagenesis, leading to gene silencing or oncogene activation. In addition, GMP-grade viral vector manufacturing can be expensive with lengthy wait times for new batches. Here we have developed a virus-free strategy in primary T cells that has eliminated the use of viral vectors through the use of CRISPR-Cas9 to precisely edit the chimeric antigen receptor into the TRAC gene1. Our method of virus free production begins through the generation of a double stranded DNA (dsDNA) template produced by polymerase chain reaction (PCR). This template is then combined with a SpCas9-single guide RNA to create a ribonucleoprotein (RNP) complex. Isolated human primary T cells from adult healthy donors are then nucleofected with the RNP and dsDNA template on day 2 of ex vivo expansion. Flow cytometry is then utilized to immunophenotype the cell product and analyze the percent of efficiency of CAR gene transfer. Within the cell product, the editing efficiencies are \u3e95% TCR knockout and 35% CAR+. Transcriptional profiling indicates that the virus-free CART cells have a favorable memory-like phenotype. In addition to our in vitro work, in vivo mice studies with anti-GD2 CART products demonstrate regression of GD2+ solid tumors upon virus-free CART treatment, showing similar potency and survival to viral-produced CAR T cells. The production of virus-free CAR T cells has high potential to enable the rapid and flexible manufacturing of highly defined and highly potent CAR T cell products for the treatment of solid tumors. 1 Mueller, K. et al. CRISPR-mediated insertion of a chimeric antigen receptor produces nonviral T cell products capable of inducing solid tumor regression. bioRxiv preprint doi: https://doi.org/10.1101/2021.08.06.455489 (2021)

Virus-Free CRISPR CAR T cells induce solid tumor regression

Chimeric antigen receptor (CAR) T cell therapy has shown promising efficacy in treating hematologic malignancies and has led to the FDA-approval of three CAR T cell products. However, there has been little success in treating solid tumors, as clinical trials to date have yielded little to no responses and no improvement in survival. Current methods of CAR T cell production typically involve the use of viral vectors which can give rise to complications such as insertional mutagenesis, leading to gene silencing or oncogene activation. In addition, GMP-grade viral vector manufacturing can be expensive with lengthy wait times for new batches. Here we have developed a virus-free strategy in primary T cells that has eliminated the use of viral vectors through the use of CRISPR-Cas9 to precisely edit the chimeric antigen receptor into the TRAC gene1. Our method of virus free production begins through the generation of a double stranded DNA (dsDNA) template produced by polymerase chain reaction (PCR). This template is then combined with a SpCas9-single guide RNA to create a ribonucleoprotein (RNP) complex. Isolated human primary T cells from adult healthy donors are then nucleofected with the RNP and dsDNA template on day 2 of ex vivo expansion. Flow cytometry is then utilized to immunophenotype the cell product and analyze the percent of efficiency of CAR gene transfer. Within the cell product, the editing efficiencies are \u3e95% TCR knockout and 35% CAR+. Transcriptional profiling indicates that the virus-free CART cells have a favorable memory-like phenotype. In addition to our in vitro work, in vivo mice studies with anti-GD2 CART products demonstrate regression of GD2+ solid tumors upon virus-free CART treatment, showing similar potency and survival to viral-produced CAR T cells. The production of virus-free CAR T cells has high potential to enable the rapid and flexible manufacturing of highly defined and highly potent CAR T cell products for the treatment of solid tumors. 1 Mueller, K. et al. CRISPR-mediated insertion of a chimeric antigen receptor produces nonviral T cell products capable of inducing solid tumor regression. bioRxiv preprint doi: https://doi.org/10.1101/2021.08.06.455489 (2021)

Efficient In Vivo Genome Editing Using RNA-Guided Nucleases Nature Biotechnology

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have evolved in bacteria and archaea as a defense mechanism to silence foreign nucleic acids of viruses and plasmids. Recent work has shown that bacterial type II CRISPR systems can be adapted to create guide RNAs (gRNAs) capable of directing site-specific DNA cleavage by the Cas9 nuclease in vitro. Here we show that this system can function in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies comparable to those obtained using ZFNs and TALENs for the same genes. RNA-guided nucleases robustly enabled genome editing at 9 of 11 different sites tested, including two for which TALENs previously failed to induce alterations. These results demonstrate that programmable CRISPR/Cas systems provide a simple, rapid, and highly scalable method for altering genes in vivo, opening the door to using RNAguided nucleases for genome editing in a wide range of organisms. Bacteria and archaea have evolved an elegant adaptive defense mechanism which uses clustered regularly interspaced short palindromic repeats (CRISPR), together with CRISPRassociated (Cas) proteins, to provide acquired resistance to invading viruses and plasmids 1-3 . The type II CRISPR/Cas system relies on uptake of foreign DNA fragments into CRISPR loci 4 and subsequent transcription and processing of these CRISPR repeatspacer arrays into short CRISPR RNAs (crRNAs) 5 , which in turn anneal to a transactivating crRNA (tracrRNA) and direct sequence-specific silencing of foreign nucleic acid by Cas proteins 5-7