Efficient and Allele-Specific Genome Editing of Disease Loci in Human iPSCs

© The American Society of Gene & Cell Therapy

original article

Efficient and Allele-Specific Genome Editing of Disease Loci in Human iPSCs

Cory Smith1,2,3, Leire Abalde-Atristain1,2,4, Chaoxia He1,2, Brett R Brodsky1, Evan M Braunstein1, Pooja Chaudhari4,5, Yoon-Young Jang2,4,5, Linzhao Cheng1,2,3,4 and Zhaohui Ye1,2

Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; 2Institute for Cell Engineer-ing, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; 3Predoctoral Training Program in Human Genetics, Johns Hopkins Univer-sity School of Medicine, Baltimore, Maryland, USA; 4Cellular and Molecular Medicine Graduate Program, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; 5Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

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Efficient and precise genome editing is crucial for realiz-ing the full research and therapeutic potential of human induced pluripotent stem cells (iPSCs). Engineered nucle-ases including CRISPR/Cas9 and transcription activator like effector nucleases (TALENs) provide powerful tools for enhancing gene-targeting efficiency. In this study, we inves-tigated the relative efficiencies of CRISPR/Cas9 and TALENs in human iPSC lines for inducing both homologous donor-based precise genome editing and nonhomologous end joining (NHEJ)-mediated gene disruption. Significantly higher frequencies of NHEJ-mediated insertions/deletions were detected at several endogenous loci using CRISPR/Cas9 than using TALENs, especially at nonexpressed tar-gets in iPSCs. In contrast, comparable efficiencies of inducing homologous donor-based genome editing were observed at disease-associated loci in iPSCs. In addition, we investigated the specificity of guide RNAs used in the CRISPR/Cas9 system in targeting disease-associated point mutations in patient-specific iPSCs. Using myeloprolifera-tive neoplasm patient-derived iPSCs that carry an acquired JAK2-V617F point mutation and α1-antitrypsin (AAT) defi-ciency patient-derived iPSCs that carry an inherited Z-AAT point mutation, we demonstrate that Cas9 can specifically target either the mutant or the wild-type allele with little disruption at the other allele differing by a single nucleo-tide. Overall, our results demonstrate the advantages of the CRISPR/Cas9 system in allele-specific genome target-ing and in NHEJ-mediated gene disruption.

Received 17 August 2014; accepted 17 November 2014; advance online publication 16 December 2014. doi:10.1038/mt.2014.226

INTRODUCTION

Techniques to edit genomic DNA at a precise locus in human induced pluripotent stem cells (iPSCs) present an unprecedented potential for regenerative medicine as well as disease modeling of genetic variants. The efficiency of multiple published tech-niques in human iPSCs has been extremely low until the devel-opment of engineered nucleases such as zinc finger nucleases

(ZFNs), transcription activator like effector nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas systems.1–6 ZFNs and TALENs are fusion proteins, in which Fok1 nuclease domain is fused to a DNA binding domain that can bind to and cleave a specific DNA sequence of interest. Once a targeted double strand break (DSB) has been introduced, the DNA is repaired by the cell’s endogenous DNA repair machin-ery through one of two pathways. The error-prone nonhomolo-gous end joining (NHEJ) pathway often results in small insertions or deletions (indels), while the Homology-directed Repair (HDR) pathway results in precise repair with a homologous chromosome or an exogenous donor template. These engineered proteins act-ing as designer nucleases proved to be an efficient means to target and manipulate the genome for both gene knock out (KO) and knock in (KI) experiments. Compared to ZFNs, design of a pair of TALENs is more feasible for most laboratories and favored by many investigators. However, a pair of TALENs, each has ~15 peptide modules of 33-amino-acid units, still takes time to syn-thesize and test to ensure its efficiency as well as specificity.

In comparison to a pair of TALENs, the CRISPR-Cas type II is more user-friendly as the protein component (Cas9) remains the same while the short RNA components for one or multiple targets can be rapidly designed and synthesized. The system was originally identified to have three essential components: (i) Cas9 endonuclease, (ii) CRISPR RNA (crRNA) to bind the complementary DNA target, and (iii) trans-activating RNA (traRNA) to associate crRNA to Cas9. This was further reduced to two components by fusing the traRNA and crRNA to a single guide RNA (gRNA).7 Cas9 has been adapted for better expression in mammalian cells and was shown to be an efficient and adaptable tool in human cell lines including iPSCs.4,5

In order to gain a better understanding of the advantages and disadvantages of CRISPR/Cas9 and TALENs technologies in human iPSCs, we compared the efficiency of Cas9-gRNAs versus TALENs in targeting disease-associated loci. We investigated their efficiencies by measuring both NHEJ-mediated indel mutations and homologous donor-based precise gene editing. The model disease genes we used include JAK2, in which an acquired somatic point mutation (JAK2-V617F) occurs in approximately 95% of patients with polycythemia vera (PV),8 and the SERPINA1 gene,

Correspondence: Zhaohui Ye, Ross Research Building, Room 1032, 720 Rutland Ave., Baltimore, Maryland 21205, USA. E-mail: zye@jhmi.edu or Linzhao Cheng, Edward Miller Research Building 747, 733 North Broadway, Baltimore, Maryland 21205, USA. E-mail: lcheng2@jhmi.eduMolecular Therapy

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