Technical considerations for the use of CRISPR/Cas9 in hematology research
2017; Elsevier BV; Volume: 54; Linguagem: Inglês
10.1016/j.exphem.2017.07.006
ISSN1873-2399
AutoresMichael C. Gundry, Daniel P. Dever, David Yudovich, Daniel E. Bauer, Simon Haas, Adam C. Wilkinson, Sofie Singbrant,
Tópico(s)Evolution and Genetic Dynamics
Resumo•Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 is a powerful tool for genome editing in hematopoietic cells, including primary hematopoietic stem and progenitor cells (HPSCs).•CRISPR/Cas9 can be used for gene disruption, gene targeting, and/or genomic screening.•Several technical aspects of the CRISPR/Cas9 toolkit, particularly single-guide RNA design and Cas9 delivery, should be considered during assay optimization. The hematopoietic system is responsible for transporting oxygen and nutrients, fighting infections, and repairing tissue damage. Hematopoietic system dysfunction therefore causes a range of serious health consequences. Lifelong hematopoiesis is maintained by repopulating multipotent hematopoietic stem cells (HSCs) that replenish shorter-lived, mature blood cell types. A prokaryotic mechanism of immunity, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system, has been recently “repurposed” to mutate mammalian genomes efficiently and in a sequence-specific manner. The application of this genome-editing technology to hematology has afforded new approaches for functional genomics and even the prospect of “correcting” dysfunctional HSCs in the treatment of serious genetic hematological diseases. In this Perspective, we provide an overview of three recent CRISPR/Cas9 methods in hematology: gene disruption, gene targeting, and saturating mutagenesis. We also summarize the technical considerations and advice provided during the May 2017 International Society of Experimental Hematology New Investigator Committee webinar on the same topic. The hematopoietic system is responsible for transporting oxygen and nutrients, fighting infections, and repairing tissue damage. Hematopoietic system dysfunction therefore causes a range of serious health consequences. Lifelong hematopoiesis is maintained by repopulating multipotent hematopoietic stem cells (HSCs) that replenish shorter-lived, mature blood cell types. A prokaryotic mechanism of immunity, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system, has been recently “repurposed” to mutate mammalian genomes efficiently and in a sequence-specific manner. The application of this genome-editing technology to hematology has afforded new approaches for functional genomics and even the prospect of “correcting” dysfunctional HSCs in the treatment of serious genetic hematological diseases. In this Perspective, we provide an overview of three recent CRISPR/Cas9 methods in hematology: gene disruption, gene targeting, and saturating mutagenesis. We also summarize the technical considerations and advice provided during the May 2017 International Society of Experimental Hematology New Investigator Committee webinar on the same topic. The mammalian hematopoietic system plays an essential role in health and disease, carrying oxygen and nutrients around the body, fighting infection, and helping to repair tissue damage. A small number of multipotent hematopoietic stem cells (HSCs) are responsible for the lifelong balanced production of mature blood cells [1Eaves C.J. Hematopoietic stem cells: concepts, definitions, and the new reality.Blood. 2015; 125: 2605-2613Crossref PubMed Scopus (304) Google Scholar]. Under homeostasis, HSCs are located in the bone marrow and maintained in a long-term quiescent state [2Wilson A. Laurenti E. Oser G. et al.Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.Cell. 2008; 135: 1118-11129Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar]. However, after stress or bone marrow transplantation, hematopoietic stem and progenitor cells (HSPCs) can be activated to expand and reestablish homeostasis [1Eaves C.J. Hematopoietic stem cells: concepts, definitions, and the new reality.Blood. 2015; 125: 2605-2613Crossref PubMed Scopus (304) Google Scholar, 3Osawa M. Hanada K. Hamada H. Nakauchi H. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.Science. 1996; 273: 242-245Crossref PubMed Scopus (1694) Google Scholar, 4Haas S. Hansson J. Klimmeck D. et al.Inflammation-induced emergency megakaryopoiesis driven by hematopoietic stem cell-like megakaryocyte progenitors.Cell Stem Cell. 2015; 17: 422-434Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar]. The ability of HSCs to repopulate efficiently and drive long-term blood formation after transplantation makes them an attractive and widely used tool in many clinical settings [5Felfly H. Haddad G.G. Hematopoietic stem cells: potential new applications for translational medicine.J Stem Cells. 2014; 9: 163-197PubMed Google Scholar]. The ability to genetically modify HSCs has therefore been a long-standing desire in clinical hematology, and one that would also represent a powerful tool for basic hematological research. The field of genome editing has been recently revolutionized by the introduction of engineered nucleases, with the promise of a controlled genomic engineering approach [6Gersbach C.A. Genome engineering: the next genomic revolution.Nat Methods. 2014; 11: 1009-1011Crossref PubMed Scopus (23) Google Scholar]. Zinc finger nucleases, transcription activator-like effector-based nucleases, and in particular, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology have been developed to introduce precise genomic alterations [7Cho S.W. Kim S. Kim J.M. Kim J.S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease.Nat Biotechnol. 2013; 31: 230-232Crossref PubMed Scopus (1315) Google Scholar, 8Cong L. Ran F.A. Cox D. et al.Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (9338) Google Scholar, 9Mali P. Yang L. Esvelt K.M. et al.RNA-guided human genome engineering via Cas9.Science. 2013; 339: 823-826Crossref PubMed Scopus (6073) Google Scholar, 10Hsu P.D. Lander E.S. Zhang F. Development and applications of CRISPR-Cas9 for genome engineering.Cell. 2014; 157: 1262-1278Abstract Full Text Full Text PDF PubMed Scopus (3388) Google Scholar, 11Sander J.D. Joung J.K. CRISPR-Cas systems for editing, regulating and targeting genomes.Nat Biotechnol. 2014; 32: 347-355Crossref PubMed Scopus (2022) Google Scholar, 12Porteus M.H. Cathomen T. Weitzman M.D. Baltimore D. Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks.Mol Cell Biol. 2003; 23: 3558-3565Crossref PubMed Scopus (132) Google Scholar]. The CRISPR/Cas9 technology was adapted from a prokaryotic immune system and includes, among others, the endonuclease Cas9 and a single-guide RNA (sgRNA), which targets the Cas9 in a desired region of genome through Watson–Crick base pairing (Fig. 1). CRISPR/Cas9-induced DNA double-strand breaks can be repaired by the error-prone non-homologous end-joining (NHEJ) pathway, which frequently results in the introduction of insertions or deletions (indels). Alternatively, homologous recombination (HR) can be exploited to introduce precise genomic modifications using homologous DNA donor templates. The field of hematology has made great progress with CRISPR/Cas9 applications, and although studies describing efficient protocols for gene disruption and HR-mediated gene-targeting approaches in various hematopoietic cell types have been published [13Gundry M.C. Brunetti L. Lin A. et al.Highly efficient genome editing of murine and human hematopoietic progenitor cells by CRISPR/Cas9.Cell Rep. 2016; 17: 1453-1461Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 14Dever D.P. Bak R.O. Reinisch A. et al.CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells.Nature. 2016; 539: 384-389Crossref PubMed Scopus (474) Google Scholar, 15Canver M.C. Smith E.C. Sher F. et al.BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis.Nature. 2015; 527: 192-197Crossref PubMed Scopus (503) Google Scholar, 16Mandal P.K. Ferreira L.M. Collins R. et al.Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9.Cell Stem Cell. 2014; 15: 643-652Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 17Tzelepis K. Koike-Yusa H. De Braekeleer E. et al.A CRISPR dropout screen identifies genetic vulnerabilities and therapeutic targets in acute myeloid leukemia.Cell Rep. 2016; 17: 1193-1205Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 18Heckl D. Kowalczyk M.S. Yudovich D. et al.Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing.Nat Biotechnol. 2014; 32: 941-946Crossref PubMed Scopus (341) Google Scholar], no reviews describe the technical considerations for the use of CRISPR/Cas9 in hematology research. In May 2017, Daniel E. Bauer, Michael C. Gundry, and Daniel P. Dever presented in an International Society ofExperimental Hematology (ISEH) webinar organized by the New Investigator Committee and moderated by David Yudovich. This webinar focused on the technical considerations for the use of CRISPR/Cas9 genome editing in hematology research. During the webinar, three methods were covered: CRISPR/Cas9 for gene disruption, CRISPR/Cas9 for gene targeting, and CRISPR/Cas9 for saturating mutagenesis. This Perspective provides a synthesis of the topics covered during the webinar, including protocols summarizing the technical advice for designing, troubleshooting, and optimizing these CRISPR/Cas9 applications. In writing it, we aim to provide the hematology research community with a useful reference for reproducing experimental CRISPR/Cas9 methodologies. Before starting CRISPR/Cas9 experiments, there are several important technical aspects to consider. In particular, the design of sgRNAs, the choice of Cas9/sgRNA reagents, and the delivery mechanism should be considered, as outlined below. There is a wide variety of web-based bioinformatics tools available for sgRNA design, and each has a slightly different algorithm for scoring on-target efficiency and specificity. For example, CRISPRscan [19Moreno-Mateos M.A. Vejnar C.E. Beaudoin J.D. et al.CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo.Nat Methods. 2015; 12: 982-988Crossref PubMed Scopus (590) Google Scholar] has UCSC browser track functionality, which can be useful in visualizing target exons/regions. In addition, the scoring system for CRISPRscan takes into account that sgRNAs transcribed from a T7 promoter (necessary for efficient in vitro transcription) must begin with two 5′ guanine nucleotides before the start of the target transcript. Mismatches between the target locus and these two 5′ guanine nucleotides can lead to variable cleavage efficiencies. There are a range of other similar bioinformatics tools available such as CHOPCHOP [20Labun K. Montague T.G. Gagnon J.A. Thyme S.B. Valen E. CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering.Nucleic Acids Res. 2016; 44: W272-W276Crossref PubMed Scopus (444) Google Scholar] and CRISPOR [21Haeussler M. Schönig K. Eckert H. et al.Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR.Genome Biol. 2016; 17: 148Crossref PubMed Scopus (787) Google Scholar]. When planning an experiment to test for the consequence of the loss-of-function of a gene, it is important to target the appropriate exons. Conserved exons can be identified using RNA-sequencing data from the cell type of interest (or the closest available cell type). Exons that are included in all transcripts should be used for sgRNA targeting. It is also best to target early exons, because mutation of later exons may not result in nonsense-mediated decay [22Popp M.W. Maquat L.E. Leveraging rules of nonsense-mediated mRNA decay for genome engineering and personalized medicine.Cell. 2016; 165: 1319-1322Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar]. In addition to these guidelines, confirmation of protein changes by Western blotting and/or flow cytometry after gene targeting is also recommended. It is important to have accurate genomic sequences for the cell lines used. Unknown single nucleotide polymorphisms (SNPs), indels, and other variants can alter existing protospacer adjacent motifs (PAMs), change the sgRNA spacer sequence, and/or generate new PAMs in a target sample. If targeting repetitive elements, there may be many off-target sites within the genome, which can complicate any downstream analysis and lead to cellular toxicity [15Canver M.C. Smith E.C. Sher F. et al.BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis.Nature. 2015; 527: 192-197Crossref PubMed Scopus (503) Google Scholar, 23Munoz D.M. Cassiani P.J. Li L. et al.CRISPR screens provide a comprehensive assessment of cancer vulnerabilities but generate false-positive hits for highly amplified genomic regions.Cancer Discov. 2016; 6: 900-913Crossref PubMed Scopus (201) Google Scholar, 24Aguirre A.J. Meyers R.M. Weir B.A. et al.Genomic copy number dictates a gene-independent cell response to CRISPR/Cas9 targeting.Cancer Discov. 2016; 6: 914-929Crossref PubMed Scopus (300) Google Scholar]. When planning a gene disruption experiment, testing two to three exons per gene with three to five sgRNAs per exon is recommended. For gene disruption experiments, sgRNAs can be combined for the same exon into a single electroporation. This often results in co-deletions between sgRNAs [13Gundry M.C. Brunetti L. Lin A. et al.Highly efficient genome editing of murine and human hematopoietic progenitor cells by CRISPR/Cas9.Cell Rep. 2016; 17: 1453-1461Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar]. Notably, the hands-on time required to make 10–20 sgRNAs is only marginally greater than that required to make a single sgRNA. When knocking out a gene in primary samples (where material is limited), first screen sgRNAs in representative cell lines (K562 cells or HL-60 cells are commonly used) to select sgRNAs with the highest gene-disruption efficiency for use with primary samples. The early protocols available for CRISPR/Cas9 involved either plasmid-based delivery through transfection or lentiviral-based delivery of Cas9 and sgRNA (Fig. 1) [6Gersbach C.A. Genome engineering: the next genomic revolution.Nat Methods. 2014; 11: 1009-1011Crossref PubMed Scopus (23) Google Scholar]. These protocols remain common for CRISPR/Cas9 screening approaches, including saturating mutagenesis. However, very low efficiency rates were seen when using primary human HSPCs [13Gundry M.C. Brunetti L. Lin A. et al.Highly efficient genome editing of murine and human hematopoietic progenitor cells by CRISPR/Cas9.Cell Rep. 2016; 17: 1453-1461Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 16Mandal P.K. Ferreira L.M. Collins R. et al.Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9.Cell Stem Cell. 2014; 15: 643-652Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar]. Ribonucleoprotein (RNP)-based delivery provides an alternative delivery approach for CRISPR/Cas9 (Fig. 1). In this strategy, the biologically active components Cas9 protein and sgRNA are complexed and then delivered directly into target cells. Allelic disruption rates exceeding 85% have been observed in both hematopoietic cell lines and primary HSPCs in the absence of selection. Importantly, the RNP method may minimize off-target effects, because it delivers only a brief pulse of Cas9 and eliminates the use of exogenous DNA, which could integrate randomly into the target cell genome. Recombinant Cas9 protein can be purchased from various vendors (e.g., Thermo Scientific, IDT). sgRNAs can be in vitro transcribed (IVT) in-house [19Moreno-Mateos M.A. Vejnar C.E. Beaudoin J.D. et al.CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo.Nat Methods. 2015; 12: 982-988Crossref PubMed Scopus (590) Google Scholar] or synthesized by various vendors (e.g., TriLink, Synthego). For the latter, the use of chemically modified synthetic RNAs is recommended [25Hendel A. Bak R.O. Clark J.T. et al.Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells.Nat Biotechnol. 2015; 33: 985-989Crossref PubMed Scopus (599) Google Scholar]. Although lentiviral constructs can be used to deliver Cas9/sgRNA, electroporation is commonly used for RNP (and plasmid) Cas9/sgRNA delivery. The most frequently used devices for delivery of RNPs into HSPCs are the Invitrogen Neon instrument (program: 1600V, 10 ms, 3 pulses), the Lonza Nucleofector 4D (program: DZ100), and the Lonza Nucleofector IIb (program: U-014). All devices are capable of allelic disruption rates exceeding 85% when optimized (although maximal efficiency is target specific). Having considered how to design sgRNAs, the types of Cas9 and sgRNA available to use, and the specialized equipment necessary for these experiments, we next outline the basic protocols for use of CRISPR/Cas9 in gene disruption, gene targeting, and saturating mutagenesis. Rather than specific point-by-point guides, these protocol focus on general advice for each step. Specific experimental details are described elsewhere [13Gundry M.C. Brunetti L. Lin A. et al.Highly efficient genome editing of murine and human hematopoietic progenitor cells by CRISPR/Cas9.Cell Rep. 2016; 17: 1453-1461Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 14Dever D.P. Bak R.O. Reinisch A. et al.CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells.Nature. 2016; 539: 384-389Crossref PubMed Scopus (474) Google Scholar, 15Canver M.C. Smith E.C. Sher F. et al.BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis.Nature. 2015; 527: 192-197Crossref PubMed Scopus (503) Google Scholar]. CRISPR/Cas9 editing can be used to establish relationships between the absence of specific gene products and the resulting changes in cellular function and structure. The following RNP-based protocol affords efficient gene disruption in primary HSPCs (or cell lines) and can be completed in just 3 days [13Gundry M.C. Brunetti L. Lin A. et al.Highly efficient genome editing of murine and human hematopoietic progenitor cells by CRISPR/Cas9.Cell Rep. 2016; 17: 1453-1461Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 26DeWitt M.A. Magis W. Bray N.L. et al.Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells.Sci Transl Med. 2016; 8: 360ra134Crossref PubMed Scopus (272) Google Scholar, 27DeWitt M.A. Corn J.E. Carroll D. Genome editing via delivery of Cas9 ribonucleoprotein.Methods. 2017; 121–2: 9-15Crossref Scopus (77) Google Scholar]. When working with primary human HSPCs, short-term preculture in the presence of cytokines is recommended to increase the gene disruption efficiency. A 36–48 hours, preculture is optimal before electroporation. Starting cell densities of 2.5 × 105 cells/mL in StemSpan II media (STEMCELL Technologies) supplemented with 100 ng/mL hSCF, 100 ng/mL hTPO, and 100 ng/mL hFLT3L is recommended. After choosing the target sequences, custom sgRNA primers are ordered; the primers contain a T7 promoter, the target sequence, and the first 15 bp of the sgRNA scaffold sequence. These primers are used in a six-cycle overlap PCR performed using a high-fidelity polymerase and a universal scaffold reverse primer [13Gundry M.C. Brunetti L. Lin A. et al.Highly efficient genome editing of murine and human hematopoietic progenitor cells by CRISPR/Cas9.Cell Rep. 2016; 17: 1453-1461Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar]. The resulting dsDNA templates are then purified and used for IVT for 4–12 hours. The IVT sgRNAs are then purified and incubated with Cas9 protein at a molar ratio of 2.5:1 for 15 minutes at room temperature to form the gene-specific RNPs. HSPCs or cell lines are electroporated in the presence of RNPs using Invitrogen Neon or Lonza Nucleofectors. After electroporation, a 24-hour recovery is used before assessing knockout efficiency. A number of assays can be used for estimating indel frequencies, although PCR of the target locus followed by amplicon sequencing of the PCR products to obtain accurate estimates of indel rates is recommended [28Hendel A. Fine E.J. Bao G. Porteus M.H. Quantifying on- and off-target genome editing.Trends Biotechnol. 2015; 33: 132-140Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 29Brinkman E.K. Chen T. Amendola M. van Steensel B. Easy quantitative assessment of genome editing by sequence trace decomposition.Nucleic Acids Res. 2014; 42: e168Crossref PubMed Scopus (1028) Google Scholar]. Amplicon sequencing libraries can be spiked into larger high-throughput sequencing runs at nominal cost [13Gundry M.C. Brunetti L. Lin A. et al.Highly efficient genome editing of murine and human hematopoietic progenitor cells by CRISPR/Cas9.Cell Rep. 2016; 17: 1453-1461Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar]. After gene disruption, the next step is often in vivo or in vitro assays to study the consequences of gene loss of function. After electroporation, the cells can be cultured further or used for in vivo experiments (e.g., transplantation into NOD SCID gamma [NSG] mice). It is of vital importance to use the appropriate controls. IVT sgRNAs can be toxic, particularly to primary cells. Using several control sgRNAs is recommended, including a cell surface marker expressed on the cell and a second target gene not expressed by the cells. Using the CRISPR/Cas9 RNP-based gene disruption approach described above, a targeted editing frequency of more than 85% in human CD34+ HSPCs can be achieved [13Gundry M.C. Brunetti L. Lin A. et al.Highly efficient genome editing of murine and human hematopoietic progenitor cells by CRISPR/Cas9.Cell Rep. 2016; 17: 1453-1461Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar]. Electroporation enables robust delivery of the RNP to virtually all CD34+ HSPCs. Furthermore, the short half-life of RNP minimizes off-target cleavage and limits HSPC toxicity: RNP-treated HSPCs did not show reduction in methylcellulose clonogenic capacity or cell viability [13Gundry M.C. Brunetti L. Lin A. et al.Highly efficient genome editing of murine and human hematopoietic progenitor cells by CRISPR/Cas9.Cell Rep. 2016; 17: 1453-1461Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar]. This method opens the door for cost-effective generation of genetic models, with quick turnaround times from biologic question to efficient CRISPR/Cas9 perturbation. Unlike the method described above, which relies on random indels via NHEJ, CRISPR/Cas9 can be used for targeted genome editing via HR. Here, a homologous DNA repair construct must be used as a template to introduce changes in the DNA. Although plasmid or linear DNA can be used, the adeno-associated virus serotype six (AAV6) system provides an efficient template, particularly for primary human HSPCs. Its use in CRISPR/Cas9-mediated HR in human HSPCs is outlined below [14Dever D.P. Bak R.O. Reinisch A. et al.CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells.Nature. 2016; 539: 384-389Crossref PubMed Scopus (474) Google Scholar]. Please note that this protocol does not cover AAV donor design or production, which have been described in detail elsewhere [30Khan I.F. Hirata R.K. Russell D.W. AAV-mediated gene targeting methods for human cells.Nat Protoc. 2011; 6: 482-501Crossref PubMed Scopus (132) Google Scholar, 31Grieger J.C. Choi V.W. Samulski R.J. Production and characterization of adeno-associated viral vectors.Nat Protoc. 2006; 1: 1412-1428Crossref PubMed Scopus (379) Google Scholar, 32Rago C. Vogelstein B. Bunz F. Genetic knockouts and knockins in human somatic cells.Nat Protoc. 2007; 2: 2734-2746Crossref PubMed Scopus (97) Google Scholar]. Alternatively, single-stranded DNA oligonucleotides donor templates may be used; although they are potentially of lower efficiency, they may be easier to synthesize [27DeWitt M.A. Corn J.E. Carroll D. Genome editing via delivery of Cas9 ribonucleoprotein.Methods. 2017; 121–2: 9-15Crossref Scopus (77) Google Scholar]. As above, it is recommended to culture the HSPCs for 36–48 hours before gene targeting to promote growth and cell viability, which enables high levels of HR. After preculture, HSPCs should be more than 80% viable and should have expanded up to threefold (depending on the HSPC source). Use of chemically modified synthetic sgRNAs is recommended for this protocol. RNP formation can be achieved by mixing sgRNA and Cas9 in a 2.5:1 molar ratio, followed by a 10-minute incubation at room temperature. However, it is recommended to determine the optimal Cas9/sgRNA ratio empirically for each target. In addition, high enough concentrations of Cas9 and sgRNA should be used to avoid taking up more than 20% of the final electroporation volume. Nucleofection is recommended for electroporating human HSPCs in this protocol. When using nucleofection cuvettes, electroporation of 0.5–1 × 106 HSPCs is optimal. Inclusion of the appropriate controls is important and should include: a mock electroporated sample (receiving no AAV or RNP), a mock electroporated sample receiving AAV, RNP-electroporated cells receiving no AAV (RNP-only), and an RNP-electroporated sample receiving AAV (RNP + AAV). The HSPCs should be resuspended in nucleofection solution, gently mixed with the RNP, and then electroporated. After electroporation, HSPCs should be diluted to 2.5–5 × 105 cells/mL with medium. Ten to 20% of the sample should be saved for the RNP-only control. Recombinant AAV6 (rAAV6) should be added to the medium immediately along with RNP-electroporated HSPCs and incubated for 12–24 hours. The time course for indel and HR frequencies may be performed between days 1 and 4 after targeting. Clonal gene targeting in hematopoietic progenitors can be tested after methylcellulose colony-forming assays. This assay can be used to evaluate the influence of gene targeting on progenitor function. Where green fluorescent protein (GFP) or another cellular marker is inserted by HR, efficiencies within phenotypically defined HSPC populations can be assessed by flow cytometry. However, the gold standard for assessing gene-targeting frequencies in HSCs is transplantation into immunocompromised NSG mice. Bulk-targeted and/or HR-enriched HSPCs should be transplanted into the femur or tail vein of sublethally irradiated 6- to 8-week-old NSG mice, and human chimerism in the bone marrow analyzed at 16 weeks after transplantation. Secondary transplantations are recommended to truly assess self-renewal capacity and long-term multilineage repopulation of gene-targeted human HSCs. For further details, see Park et al. [33Park C.Y. Majeti R. Weissman I.L. In vivo evaluation of human hematopoiesis through xenotransplantation of purified hematopoietic stem cells from umbilical cord blood.Nat Protoc. 2008; 3: 1932-1940Crossref PubMed Scopus (39) Google Scholar]. The HR-based repair in HSPCs is a potentially transformative approach for both basic and clinical applications. Remarkably, this CRISPR/Cas9-mediated HR protocol can achieve efficiencies of more than 20% of primary human CD34+ HSPCs. Significantly, within AAV6-transduced colony-forming HSPCs, more than 90% of the cells underwent a single HR repair, of which more than 40% achieved homozygous modification [14Dever D.P. Bak R.O. Reinisch A. et al.CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells.Nature. 2016; 539: 384-389Crossref PubMed Scopus (474) Google Scholar]. Whereas gene correction is likely the most clinically relevant application of this method, various types of HR donor designs are possible, including a gene insertion knock-in, a knock-in reporter cassette for precise gene disruption, or an SNP-type modification for amino acid substitution. Combined, this approach has far-reaching implications both for studying precise genetic alterations in disease modeling and the clinical setting of “gene-correcting” hereditary diseases. Although the methods described above use CRISPR/Cas9 to disrupt or repair gene function, the role of noncoding regions of the genome can also be interrogated with similar methods. By tiling sgRNAs throughout a region of interest, it is possible to achieve saturating mutagenesis screening [15Canver M.C. Smith E.C. Sher F. et al.BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis.Nature. 2015; 527: 192-197Crossref PubMed Scopus (503) Google Scholar, 34Canver M.C. Lessard S. Pinello L. et al.Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci.Nat Genet. 2017; 49: 625-634Crossref PubMed Scopus (67) Google Scholar]. This approach has been applied recently to the study of noncoding regions that determine globin gene expression [15Canver M.C. Smith E.C. Sher F. et al.BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis.Nature. 2015; 527: 192-197Crossref PubMed Scopus (503) Google Scholar]. Given the large numbers of cells needed for such screening, this approach is most easily undertaken using cell lines, preferably one that is already engineered to express Cas9 constitutively. All possible 20 bp sgRNA target sequences upstream of PAM (or other recognition) sites should be identified within the genomic region of interest. Importantly, the PAM restriction of the nuclease used will determine the density of mutagenesis possible within the region of interest. DNA oligos for these sgRNA sequences can be ordered as a pool and cloned via Gibson Assembly into a lentivirus backbone. Previous studies have use
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