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      A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells

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          Abstract

          Translation of the CRISPR/Cas9 system to human therapeutics holds high promise. Specificity remains a concern, however, especially when modifying stem cell populations. We show that existing rationally-engineered Cas9 high fidelity variants have reduced on-target activity using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, R691A (HiFi Cas9), retained high on-target activity while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically-relevant loci ( HBB, IL2RG, CCR5, HEXB, TRAC) in human CD34 + hematopoietic stem and progenitor cells (HSPCs) as well as primary T-cells. We also show that the HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing Glu6Val mutation in SCD patient derived HSPCs. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome editing applications.

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          Most cited references45

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          FLASH: fast length adjustment of short reads to improve genome assemblies.

          Next-generation sequencing technologies generate very large numbers of short reads. Even with very deep genome coverage, short read lengths cause problems in de novo assemblies. The use of paired-end libraries with a fragment size shorter than twice the read length provides an opportunity to generate much longer reads by overlapping and merging read pairs before assembling a genome. We present FLASH, a fast computational tool to extend the length of short reads by overlapping paired-end reads from fragment libraries that are sufficiently short. We tested the correctness of the tool on one million simulated read pairs, and we then applied it as a pre-processor for genome assemblies of Illumina reads from the bacterium Staphylococcus aureus and human chromosome 14. FLASH correctly extended and merged reads >99% of the time on simulated reads with an error rate of <1%. With adequately set parameters, FLASH correctly merged reads over 90% of the time even when the reads contained up to 5% errors. When FLASH was used to extend reads prior to assembly, the resulting assemblies had substantially greater N50 lengths for both contigs and scaffolds. The FLASH system is implemented in C and is freely available as open-source code at http://www.cbcb.umd.edu/software/flash. t.magoc@gmail.com.
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            Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells.

            CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34(+) hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.
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              CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells.

              The β-haemoglobinopathies, such as sickle cell disease and β-thalassaemia, are caused by mutations in the β-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure β-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult β-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for β-haemoglobinopathies.
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                Author and article information

                Journal
                9502015
                8791
                Nat Med
                Nat. Med.
                Nature medicine
                1078-8956
                1546-170X
                27 June 2018
                06 August 2018
                August 2018
                06 February 2019
                : 24
                : 8
                : 1216-1224
                Affiliations
                [a ]Integrated DNA Technologies, Inc., Coralville, IA 52241, USA
                [b ]Department of Pediatrics, Stanford University, Stanford, CA 94305
                [c ]Department of Bioengineering, Rice University, Houston, TX 77030, USA
                [d ]Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark
                [e ]Aarhus Institute of Advanced Studies (AIAS), Aarhus University, Høegh-Guldbergs Gade 6B, Aarhus C, Denmark
                [f ]Biomaterials and Advanced Drug Delivery Laboratory, Stanford University School of Medicine, Palo Alto, CA 94304, USA
                Author notes
                [* ]Co-corresponding authors: Mark A. Behlke, Integrated DNA Technologies, Inc., 1710 Commercial Park, Coralville, IA 52241 USA, mbehlke@ 123456idtdna.com , Tel: 319-626-8432; Matthew H. Porteus, Department of Pediatrics, Stanford University, Stanford, CA 94305 USA, mporteus@ 123456stanford.edu , Tel: 650-725-6520

                AUTHOR CONTRIBUTIONS

                C.A.V. performed and designed the bacterial screen and subsequent identification of the HiFi mutants. C.A.V., M.A.C., and N.M.B. cloned and purified all proteins examined in this study. C.A.V., G.R.R., R.T., A.M.J., and M.S.M. performed NGS on-target and off-target editing experiments with RNP in human cells. C.A.V., M.A.C., and S.Y. created and characterized the HEK293 Cas9 stable cell lines. D.P.D., J.C., R.O.B., V.W., M.P.-D., and N.G.-O. carried out experiments related to HSPC and T-cell gene editing. G.B., C.M.L., and S.H.P. carried out the next generation sequencing analysis of HSPC HBB editing events. W.S. carried out the HPLC analysis of hemoglobin tetramers. M.A.B. and M.H.P. directed the research and participated in the design and interpretation of the experiments and the writing of the manuscript. C.A.V., D.P.D., M.H.P., and M.A.B. wrote the manuscript with assistance from all authors.

                Article
                NIHMS977825
                10.1038/s41591-018-0137-0
                6107069
                30082871
                9291bca3-5a72-4303-8855-179e5ce655ec

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