Elsevier

Methods

Volumes 164–165, 15 July–1 August 2019, Pages 18-28
Methods

Improving homology-directed repair efficiency in human stem cells

https://doi.org/10.1016/j.ymeth.2019.06.016Get rights and content

Highlights

Abstract

The generation of induced pluripotent stem cell models of human disease requires efficient modification of one or both alleles depending on dominant or recessive inheritance of the disease. To faithfully recapitulate many disease variants, the introduction of a single base change is required. The introduction of additional silent mutations designed to prevent re-cutting of the modified allele by Cas9 is not an optimal strategy, particularly for non-coding variants. Here, we developed an improved protocol for efficient engineering of single nucleotide variants in human iPS cells. Using a fluorescent BFP->GFP assay to monitor the incorporation of a single base pair change, we optimized the protocol to achieve HDR in 70% of unselected human iPS cells. The additive effects of cold shock, a small molecule enhancer of HDR and chemically modified ssODN dramatically shift the bias of repair in favor of HDR, resulting in a seven-fold higher ratio of HDR to NHEJ from 0.5 to 3.7.

Introduction

Since the discovery in 2007 that adult somatic cells can be reprogrammed to an embryonic, pluripotent stem cell state [1], induced pluripotent stem (iPS) cells have largely replaced embryo-derived stem (ES) cells as a source of human stem cells for research. While there were initial concerns that human iPS cells were not functionally equivalent to human ES cells, a more recent carefully controlled study has demonstrated a remarkable equivalence between human ES and iPS cells derived from the same genetic background [2]. Other advances in the culture of iPS cells, particularly the development of chemically-defined media for feeder-free culture of a highly uniform population of undifferentiated iPS cells, has greatly simplified the experimental manipulation of human iPS cells. By recapitulating developmental signals that operate during early embryogenesis, efficient protocols and media have been developed to differentiate iPSCs in vitro to many cell types of the human body [3]. Thus, in contrast to immortalized human cell lines, iPS cells provide an ideal model cell to study normal cellular, biochemical and developmental processes in a stable diploid cell. Moreover, iPS cells derived from patients [4] have been used extensively to model disease processes and as a platform to test possible therapeutic drugs that may ameliorate the disease.

The CRISPR-Cas family of programmable nucleases has revolutionized our ability to engineer the genomes of virtually any eukaryotic cell or embryo [5]. The main practical advantage of CRISPR-Cas over other nucleases is their ease of use, requiring only the design of a short 20–22 nucleotide sequence in the guide RNA to direct Cas nuclease to its target. Initially demonstrated with meganucleases [6], the introduction of a double strand break (DSB) at a genomic site can be repaired via a variety of cellular DNA repair mechanisms [7]. The most frequent outcome of DSB repair is the generation of small insertions/deletions (indels) through error-prone non-homologous end-joining (NHEJ), providing a highly efficient method to disrupt genes. In the presence of an exogenous DNA repair template containing sequences homologous to the region around the DSB, it is possible to introduce precise genetic changes by making use of other, albeit less efficient, homology directed repair (HDR) pathways. Although not a natural substrate for DSB repair, single strand oligonucleotides (ssODN) are efficacious donor templates [8]. Due to their small size (<200 bases), ssODN are used to introduce small genetic changes, from single nucleotide variants (SNVs) to insertions of small protein tags [9]. Maximal editing efficiency is attained with ssODN donors above 80 bases with equal lengths of homology on either side of the DSB. However, one study suggests that higher HDR rates can be achieved using asymmetric donors with as little as 35 bases complementary to the non-target strand [10]. Two distinct mechanisms have been proposed to describe how ssODN is incorporated at the site of the double strand break; single-strand DNA incorporation (ssDI) and synthesis-dependent strand annealing (SDSA) [11]. Importantly, the distance between the double strand break and the desired variant is critical. As a general rule, base changes located outside of the CRISPR site will not be efficiently retained.

With the advent of CRISPR-Cas genome editing technology, isogenic pairs of disease and non-disease cell lines can be generated to address the specific molecular mechanisms underlying a disease [4]. However, differences in the genetic background between individual patients is a major confounding factor and the phenotypes observed in cells may not be reproducible in different patient-derived iPS cell lines carrying the same disease mutation [2]. An alternative approach is to introduce disease variants by genome editing of a reference iPS cell line(s) derived from a normal individual [12]. This strategy has the advantage of controlling the genetic background of the cell such that the phenotype of each variant for a disease can be accurately determined and allows one to compare the phenotype of different variants for a given disease gene [13].

In vitro assembled Cas9 ribonucleoprotein (RNP) is the preferred reagent for editing human iPS and can be efficiently delivered to cells by nucleofection [14]. Cas9 RNP is immediately active when the donor template is at maximal levels and is degraded within 24 h, reducing potential off-target damage by avoiding prolonged expression of an active nuclease. Engineered, high fidelity variants of Cas9 are now available commercially as recombinant protein and should further reduce off-target damage at related sites, including the modified on-target allele [15]. The identification of factors that improve genome editing is an area of intense research and highly relevant to clinical applications of CRISPR for ex vivo and in vivo editing of human cells. For example, chemical modifications that stabilize the guide RNA and ssODN in cells have been demonstrated to enhance editing [16], [17]. A number of small molecules have been reported to shift repair outcomes towards HDR either by blocking NHEJ or activating proteins involved in HDR [18], [19], [20]. Other treatments that improve rates of HDR include cell synchronization [21], cold shock of cells [22], and physical coupling of the ssODN to Cas9 RNP [23], [24], [25].

Modeling human disease requires efficient modification of one or both alleles depending on dominant or recessive inheritance. Most human diseases are caused by single nucleotide changes and thus require editing of SNV alleles, particularly in cases of non-coding variants. The introduction of additional ‘silent’ or ‘blocking’ mutations (e.g. PAM mutations) may have unintended consequences on gene expression, thus, we focused on improving the protocol for engineering ‘scarless’ SNV alleles. In our initial experiments, nucleofection of Cas9 RNP and ssODN typically yielded a few percent of cells with the desired single nucleotide variant in one or both copies of the gene. Due to the intrinsic bias favoring NHEJ over HDR, the vast majority of clones showed NHEJ damage on one or both alleles. Based on a fluorescent BFP->GFP assay, we found the combination of cold shock, the small molecule Alt-R HDR enhancer, and end-modified ssODN shifted the repair outcome overwhelmingly in favor of HDR, resulting in conversion of BFP to GFP in 70% of iPS cells. Our improved conditions significantly reduce re-cutting and damage to the modified SNV and do not appear to increase off-target damage at related sites in the genome. In this chapter, we also discuss simple steps that can be taken to minimize genetic heterogeneity between edited clones.

Section snippets

Protocol

Human iPS cell lines can be cultured indefinitely in commercially-sourced, chemically defined media and retain a normal, diploid karyotype over many passages. The properties of human iPS cells and improved culture reagents make them highly amenable to genome editing by CRISPR-Cas9. These adherent cells grow rapidly, readily take up protein and nucleic acid by nucleofection and can be seeded at single cell density, forming visible colonies within 10 days. iPS cell colonies are easy to pick and

KOLF2-C1, a reference human iPSC line

To ensure a uniform genetic background between edited clones, we established a clonal subline of early passage, factor-free iPS cells derived from a normal individual. Several early passage iPS cell lines (p6-p8) were obtained from the HipSci resource (www.hipsci.org). All iPS cell lines were derived from fibroblast biopsies of normal healthy donors using the non-integrating Sendai virus reprogramming system. Following adaptation of the cells to feeder-free culture, the cell lines were screened

Concluding remarks

We describe an improved protocol for high efficiency editing of ‘scarless’ single nucleotide variants in human iPS cells. We show that a combination of cold shock, Alt-R HDR enhancer and end-blocked, chemically-modified ssODN dramatically shifts the balance of repair events in favor of HDR. Remarkably, 70% of nucleofected cells carry the corrected GFP-positive allele without any enrichment or selection steps.

The success of scarless editing of SNVs with Cas9 is dependent on finding active guide

Declaration of Competing Interest

None.

Acknowledgements

We wish to acknowledge Miriam Reuter and Stephen Scherer (Hospital for Sick Kids, Toronto) for sharing their analysis of pathogenic variants in KOLF2 exome sequence. We also are indebted to Garrett Rettig, Mollie Schubert and Bernice Thommandru (IDT) for helpful discussions and generously providing Alt-R HDR modified ssODN for this study.

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