Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.
The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.
Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to control gene expression1,2. These elements are typically 100 to 1,000 base pairs (bp) long. Promoters and enhancers are the two most characterized types of CREs. Promoters are present in close proximity to the transcription start sites and constitute the basic unit of transcription. Many genes have more than one promoter and their alternative use contributes to transcriptome diversity and tissue specificity3,4. On the other hand, enhancers activate transcription and can be located upstream, downstream or within introns of the target genes. Enhancers can act from far distance (over a megabase) and independent of orientation1,2. CREs also include silencers and insulators5,6. The former acts oppositely to enhancers to inhibit gene expression by binding to transcriptional repressors, whereas the latter partitions the genome into discrete topologically domains to insulate genes from other CREs from neighboring domains. These elements act in concert with each other through short- and/or long-range chromatin interactions and are organized into regulatory hubs to direct proper spatiotemporal gene expression. Recent advancements in high-throughput sequencing techniques have accelerated the identification and functional annotation of many CREs that have greatly facilitated our understandings of the transcriptional networks that dictate lineage-specific gene expression in different cell and tissue types7,8,9,10,11,12.
Given the fundamental roles of CREs in regulating transcription, their alterations can lead to aberrant gene expression. It has been shown that CREs are frequently disrupted by genetic and epigenetic changes in different types of human cancers, thereby contributing to tumor initiation, progression and aggressiveness13,14. In addition, CRE-binding factors are often mutated and/or misexpressed in various cancer types, further highlighting the significance of CRE deregulation in oncogenesis15. CREs can also be affected by structural aberrations, as exemplified by frequent chromosomal rearrangements of the immunoglobulin heavy (IgH) gene enhancer that result in abnormal activation of the neighboring oncogenes in B-cell lymphomas16. In acute myeloid leukemia (AML), repositioning of a single enhancer by chromosome 3q rearrangements causes concomitant GATA2 downregulation and EVI1 activation, which can be potentially targeted by BET inhibition of enhancer functions17. Recently, we characterized a novel chromosomal translocation involving perturbation of a RUNX1 intronic silencer that might contribute to AML progression in a pediatric patient18. Thus, deciphering the non-coding cancer genome provides fruitful avenues for elucidating disease pathogenesis, biomarker discovery and therapeutic interventions, which ultimately improve patient outcomes.
The CRISPR/Cas9 nuclease pathway, originally identified as an adaptive immune system in prokaryotic cells, has been exploited as a rapid and cost-effective means for site-specific genomic editing in living cells and organisms19,20,21,22. The CRISPR/Cas9 system involves two principal components: the gRNA and Streptococcus pyogenes-derived Cas9 nuclease. The gRNA contains a specific sequence called protospacer that recognizes the target region and directs Cas9 for editing. The gRNA is composed of two parts: CRISPR RNA (crRNA), typically a 20-mer nucleotide sequence complementary to the target DNA, and a trans-activating crRNA (tracrRNA), which serves as a binding scaffold for the nuclease. A protospacer adjacent motif (PAM) (5'-NGG) immediately adjacent to the target site is required for Cas9 cleavage and the cleavage site is located 3 nucleotides upstream of the PAM. CRISPR/Cas9-mediated gene editing is commonly performed by transfecting cells with a plasmid that encodes Cas9 and the cloned gRNA23. However, this approach is challenging for hematopoietic cells, which are often hard to transfect and require lengthy virus-based transduction methods. An alternative approach is direct cellular delivery of preassembled Cas9/gRNA RNP complexes24. A common method for the RNP delivery is electroporation, which generates temporary pores in the cell membrane, thus allowing entry of the RNP complexes into the cells25,26. The advantages of this approach include ease of use, reduced off-target effects and stability of the RNP complexes. Here, we describe a protocol of using the RNP delivery method to investigate the transcriptional role of a RUNX1 intronic silencer in the OCI-AML3 leukemia cell line18, which was established from the peripheral blood of an AML patient diagnosed with the French-American-British M4 subtype27. The protocol includes the design of crRNA, preparation of RNP complexes, electroporation as well as screening and subsequent characterization of the desired clones.
1. Design of crRNA
2. Design of Deletion Screening Primers
3. Preparation of Cas9/gRNA RNP Complexes
4. Electroporation of the RNP Complexes into OCI-AML3 Cells
5. Screening and Selection of Cell Clones with Biallelic Deletions
6. Functional Analyses of the Silencer Deletion by Real-time Quantitative RT-PCR Analysis
The aim of this experiment is to delete an intronic silencer in the RUNX1 gene and examine the impacts on RUNX1 transcription in OCI-AML3 cells. The silencer was identified by a combinatorial molecular approach and found to contain a 209 bp core element18. To enable more accurate evaluation of this core element in controlling RUNX1 expression, the crRNAs (crRNA-1 and crRNA-2) were designed to target closely to this region18 (Figure 1). The predicted Cas9 cleavage sites brought by crRNA-1 and crRNA-2 were 29 bp and 35 bp from the core element, respectively (Figure 1). It should be noted that while the PAM sites of the two crRNAs reside on opposite strands, DSBs will occur independently of PAM sequence location. Thus, the concomitant introduction of two Cas9/gRNA RNP complexes guided by crRNA-1 and crRNA-2 is expected to excise the silencer element from the RUNX1 locus.
To screen for the desired deletions in a large number of samples, a 96-well format of genomic DNA extraction kit was used for high-throughput purification. Also, DNA bound on the wells of the extraction plates can be subjected to direct amplification, thus minimizing errors or contamination due to repeated sample transfer. The primers used for screening of the deletions18 are shown in Figure 1. The expected size of the wild-type PCR product is about 500 bp. Since the intended deletion spans 273 bp, the mutant product is expected to be about 230 bp. This size range enables simple and rapid fragment analysis by capillary gel electrophoresis. A total of 160 initial cell pools were screened and 14 were found to carry the expected deletions with mutant levels of at least 70%. Five pools were then selected for further serial dilutions to identify clones bearing biallelic deletions. Representative electropherograms from cell clones with different levels of mutant products are shown in Figure 2A. The identity of the deletions was verified by Sanger sequencing (Figure 2B). As expected, indels formed by non-homologous end joining repair of DSBs33 were observed at the predicted cleavage sites in the deletion clones. These resulted in the amplification of mutant products of varying sizes, which could also be detected by capillary electrophoresis (Figure 2A).
The RUNX1 gene contains two promoters namely the distal P1 and proximal P2, which are separated by a large intron harboring the silencer element34. Three major mRNA transcripts are produced by these promoters: RUNX1c by P1 and RUNX1a and RUNX1b by P234. The nucleotide sequence of RUNX1c and RUNX1b are identical except the former has a unique N-terminus, from which a specific TaqMan gene expression assay can be designed (Figure 3A). To measure RUNX1b, a TaqMan assay recognizing both RUNX1b and RUNX1c was used (Figure 3A). RUNX1b levels were then determined by subtracting total RUNX1b/RUNX1c from RUNX1c. RUNX1a is a distinctly shorter isoform due to alternative splicing and a specific TaqMan assay is available for this variant (Figure 3A). Thus, the activity of the P1 and P2 promoters can be determined individually. Real-time quantitative RT-PCR showed that deletion of the silencer element significantly upregulated the expression levels of both P1- and P2-derived transcripts (Figure 3B).
Figure 1: Strategy to delete the RUNX1 intronic silencer. The silencer (red box) is located in the first intron of the RUNX1 gene separating the two promoters P1 and P2 (hg19 coordinates are shown). Two crRNAs (crRNA-1 and crRNA-2) were designed to introduce DSBs flanking the silencer element. The predicted Cas9 cleavage sites are indicated by vertical red lines and the PAM sites (NGG) are in purple. The two primers used for screening of the deletions are represented by open arrows. Please click here to view a larger version of this figure.
Figure 2: Identification of the deletion clones. (A) Representative electropherograms of cell clones showing different levels of mutant PCR products (MUT). The size of the mutant products varies among the clones because of indels formed at the cleavage sites. WT = wild-type. (B) Verification of the deletions from representative clones by Sanger sequencing. Please click here to view a larger version of this figure.
Figure 3: Functional consequences of the silencer deletion. (A) The three major RUNX1 isoforms (RUNX1a, RUNX1b and RUNX1c) are shown. These variants contain the same Runt DNA binding domain but different N- (orange) or C-terminus (blue). The location of the TaqMan probe/primer pairs is indicated by red lines. Numbers indicate the amino acid residues. (B) Real-time quantitative RT-PCR analysis of RUNX1 P1- and P2-derived transcripts in cell populations with (DEL) or without (WT) the biallelic deletions18. GAPDH was used for normalization. * and ** indicate PÂ < 0.05 and PÂ < 0.01, respectively by the Mann-Whitney test. This figure has been modified from Cheng et al.18. Please click here to view a larger version of this figure.
The CRISPR/Cas9 system has been used in a wide range of genome editing applications such as gene knockout and knock-in studies35,36, transcriptional regulation37,38, genetic engineering of various model organisms39,40,41,42,43,44 and gene therapy45,46. Here, we demonstrate the use of CRISPR/Cas9 to investigate the functional consequences of deleting an intronic silencer on the RUNX1 gene. The delivery of the CRISPR components in our approach did not rely on plasmid DNA, cloning of gRNA or virus but electroporation of preassembled Cas9/gRNA RNP complexes. It has been shown that the use of exogenous DNA can be associated with undesirable integration of foreign vector sequences into the host genome, increased toxicity and low efficiency25,47,48, whereas virus transduction methods are time-consuming. In addition, prolonged expression of Cas9 from plasmid DNA can augment off-target effects48. On the contrary, the direct RNP-based delivery approach has been established as the preferred method as it is fast and straightforward with improved editing efficiency, selectivity and cell viability. Indeed, a variety of methods such as lipofection49,50, electroporation25,51, nanoparticles52, cell-penetrating peptides53, iTOP54 and TRIAMF55 have been developed for efficient CRISPR/Cas9 delivery into diverse cell types as well as animal and plant species24,25,26,56,57,58,59,60,61,62,63. Since non-coding DNA sequences are hotspots of genetic variations64, checking the presence of common SNPs/indels in the target and neighboring PAM sequences is particularly relevant when designing gRNA that targets regulatory elements.
A bottleneck in CRISPR/Cas9 genome editing involves screening of desired mutant clones in a large number of samples. We employed fluorescent PCR coupled with capillary gel electrophoresis for the screening as the target mutation is a small genomic deletion of about 300 bp. This method is rapid and sensitive and can be performed in a high-throughput fashion. Also, this method allows accurate estimation of mutant levels and deletion sizes simultaneously. In addition, multiplex analysis of PCR fragments labeled with different fluorescent dyes is supported. We have been routinely using this technique to genotype small insertions/deletions in myeloid neoplasms65,66. In our experience, we can consistently detect fragment sizes that are differ by 4 bp with high precision and mutant burden down to ~3 %. However, it should be noted that this method has a fragment size limit of 1,200 bp, and thus it is not suitable for screening of large deletions. Also, base substitutions (resulting in unchanged fragment size) and potential off-target events in other genomic regions cannot be detected. For the latter, the costly whole genome sequencing is required to comprehensively profile global undesirable changes in the target clones. To adopt our current approach for investigation of large non-coding regulatory sequences (>1,000 bp), a detailed deletion and mutagenesis analyses of putative transcription factor binding sites using in vitro reporter gene assays can be performed beforehand to delineate the minimal functional region for CRISPR/Cas9 editing18.
As many genes contain more than one promoters3,4, it is important to be aware of the existence of alternative promoters in the target gene locus as manipulating regulatory elements may affect the promoters differentially. Thus, transcript variants derived from different promoters need to be measured individually to evaluate any promoter-specific responses. The use of TaqMan probe-based assays is preferred over SYBR Green because of better specificity and reproducibility. If the more advanced digital PCR system is available, transcript quantification can be performed more precisely without the need of standard curve construction.
An important consideration in performing CRISPR/Cas9 experiments in cancer cell lines is the ploidy and target gene copy number in the cells used as virtually all cancer cell lines harbor genetic alterations including structural and copy number variations. In our case, OCI-AML3 has a hyperdiploid karyotype with 45 to 50 chromosomes. Also, the cell line was found to carry a normal RUNX1 copy number as revealed from the Cancer Cell Line Encyclopedia67 and fluorescence in situ hybridization studies18. When targeting a gene with copy number gain, the delivery method may need to be optimized to provide sufficient levels of the CRISPR components for the editing. Also, more clones may need to be screened in order to identify the complete knockouts. Importantly, it has been shown that targeting at amplified genomic regions, particularly those caused by structural rearrangements, can trigger gene-independent antiproliferative responses in cancer cells, leading to false-positive results in gene functional studies68,69,70. In this regard, alternative approaches like RNA interference (RNAi) knockdown and/or cDNA overexpression should be employed to verify the CRISPR findings. Also, multiple cell lines should be used to avoid misinterpretation of cell line-specific but gene-independent CRISPR editing effects.
The CRISPR/Cas9 system has revolutionized basic and translational research by providing a simple and efficient means to genome editing. Here we demonstrate the ease of using CRISPR/Cas9 to disrupt an intronic silencer for transcriptional studies in a cancer cell line. This technique allows for the study of CREs at the DNA level and offers the opportunities to examine CRE functions in the endogenous context rather than the traditional heterologous reporter genes. Recently, a CRISPR-based RNA editing system has also been identified71 and may serve as a novel tool to study CREs by targeting RNA transcribed from the regulatory elements. By combining with chromosome conformation capture techniques, CRISPR/Cas9 will certainly help decipher the involvements of CREs in altered genome organization and gene expression linked to various health problems.
The authors would like to thank Prof. M.D. Minden (Princess Margaret Cancer Centre, University Health Network, Toronto, Canada) for providing the OCI-AML3 cell line. Also, the authors would like to thank the Core Utilities of Cancer Genomics and Pathobiology (The Chinese University of Hong Kong) for providing the facilities and assistance in support of this research.
Name | Company | Catalog Number | Comments |
0.2 cm-gap electroporation cuvette | Bio-Rad | 1652086 | |
1×TE buffer, pH7.5 | Integrated DNA Technologies | 11-01-02-02 | |
10mM dNTP mix | Thermo Fisher Scientific | 18427013 | |
3500 Genetic Analyzer | Thermo Fisher Scientific | 4405673 | |
6-FAM-labeled fluorescent PCR forward primer | Thermo Fisher Scientific | None | |
7300 Real-Time PCR System | Thermo Fisher Scientific | None | |
7300 System SDS Software | Thermo Fisher Scientific | None | Version 1.3.1 |
Bio-Rad Gene Pulser Xcell system | Bio-Rad | 1652660 | |
ChargeSwitch Direct gDNA Purification Kit, 96-well | Thermo Fisher Scientific | CS11205 | |
crRNA-1 | Integrated DNA Technologies | Alt-R CRISPR-Cas9 crRNA | Components of the Cas9/gRNA complex |
crRNA-2 | Integrated DNA Technologies | Alt-R CRISPR-Cas9 crRNA | Components of the Cas9/gRNA complex |
Deionized formamide | Thermo Fisher Scientific | 4311320 | |
Electroporation Enhancer | Integrated DNA Technologies | 1075915 | |
fetal bovine serum | Thermo Fisher Scientific | 10270098 | |
Fluorometer | Thermo Fisher Scientific | Q32857 | |
GeneMapper Software 5 | Thermo Fisher Scientific | 4475073 | |
GeneScan 600 LIZ Size Standard | Thermo Fisher Scientific | 4408399 | |
GlutaMAX | Thermo Fisher Scientific | 35050061 | |
PBS, 10X Solution, pH7.4 | Affymetrix | 75889 | |
Penicillin and streptomycin | Thermo Fisher Scientific | 15140122 | |
Platinum Taq DNA Polymerase High Fidelity | Thermo Fisher Scientific | 11304029 | |
Qubit dsDNA HS Assay Kit | Thermo Fisher Scientific | Q32854 | |
Recombinant S. pyogenes Cas9 nuclease | Integrated DNA Technologies | 1081058 | Components of the Cas9/gRNA complex |
RPMI 1640 medium | Thermo Fisher Scientific | 31800-022 | |
RPMI 1640 medium without phenol red | Thermo Fisher Scientific | 11835030 | |
RUNX1a TaqMan gene expression assays | Thermo Fisher Scientific | 4331182 | Hs04186042_m1 |
RUNX1b/c TaqMan gene expression assays | Thermo Fisher Scientific | 4331182 | Hs00231079_m1 |
RUNX1c TaqMan gene expression assays | Thermo Fisher Scientific | 4331182 | Hs01021966_m1 |
SuperScript III First-Strand Synthesis System | Thermo Fisher Scientific | 18080051 | |
TaqMan Universal PCR Master Mix | Thermo Fisher Scientific | 4304437 | |
tracrRNA | Integrated DNA Technologies | 1072533 | Components of the Cas9/gRNA complex |
TRIzol Reagent | Thermo Fisher Scientific | 15596018 | |
Unlabeled PCR reverse primer | Thermo Fisher Scientific | None |
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