Knock-In Strategy for Editing Human and Zebrafish Mitochondrial DNA

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Letter

A knock-in strategy for editing human and zebrafish mitochondrial DNA using mito-CRISPR/Cas9 system Wan-ping Bian, Yanling Chen, Juanjuan Luo, Chao Wang, Shaolin Xie, and Desheng Pei ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00411 • Publication Date (Web): 08 Apr 2019 Downloaded from http://pubs.acs.org on April 9, 2019

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ACS Synthetic Biology

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A knock-in strategy for editing human and zebrafish

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mitochondrial DNA using mito-CRISPR/Cas9 system

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Running Title: mtDNA editing based on mito-CRISPR/Cas9

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Wan-Ping Bian1†, Yan-Ling Chen1†, Juan-Juan Luo1, Chao Wang1, Shao-Lin Xie1,

7

De-Sheng Pei1*

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1Key

Laboratory of Reservoir Aquatic Environment, Chongqing Institute of Green

and Intelligent Technology, Chinese Academy of Sciences, Chongqing 400714, China

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*For

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†These

correspondence: [email protected] or [email protected] authors contributed equally to this work

ACS Paragon Plus Environment

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Abstract

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The mitochondria DNA (mtDNA) editing tool, Zinc finger nucleases (ZFNs),

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Transcription activator-like effector nuclease (TALENs), and Clustered regularly

18

interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9)

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system, is a promising approach for the treatment of mtDNA diseases by eliminating

20

mutant mitochondrial genomes. However, there have been no reports of repairing the

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mutant mtDNA with homologous recombination strategy to date. Here, we show a

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mito-CRISPR/Cas9 system that mito-Cas9 protein can specifically target mtDNA and

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reduce mtDNA copy number in both human cell and zebrafish. An exogenous

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single-stranded DNA (ssDNA) with short homologous arm was knocked into the

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targeting loci accurately, and this mutagenesis could be steadily transmitted to F1

26

generation of zebrafish. Moreover, we found some major factors involved in nuclear

27

DNA repairing were up-regulated significantly by the mito-CRISPR/Cas9 system.

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Taken together, our data suggested that the mito-CRISPR/Cas9 system could be a

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useful method to edit mtDNA by knock-in strategy, providing a potential therapy for

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the treatment of inherited mitochondrial diseases.

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KEYWORDS:

32

zebrafish

mito-CRISPR/Cas9;

mitochondria;


mtDNA

editing;

ssDNA;

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Mitochondria are the double-membrane cellular organelles that could produce

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adenosine triphosphate (ATP) through the process of the oxidative phosphorylation in

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most eukaryotes. However, besides supplying the energy, mitochondria are involved

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in other processes, such as energy metabolism, calcium storage, and regulation of cell

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death pathway1. One cell contains numerous mitochondria with dozens of copies of

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mitochondrial DNA (mtDNA) in each mitochondrion. The mtDNA encodes 13

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essential proteins involved in the oxidative phosphorylation system, 2 ribosomal

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RNAs (rRNAs), and 22 transfer RNAs (tRNAs) for translation of mtDNA-encoded 13

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mRNAs in human cell and zebrafish

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have been identified or predicted in mitochondria, the majority of them are encoded

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by nuclear genes 5. Many previous studies have shown that mtDNA point mutation is

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associated with a diverse range of inherited diseases ， which commonly affects

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different organs with the higher energy requirements, including the brain, muscles,

47

heart, and others

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co-exists with normal mtDNA in mitochondria. Once the copies of mutant mtDNA

49

exceeds a threshold, a mitochondria disease symptom may appear 7. The threshold

50

levels of mtDNA mutation are commonly from 60% to 95% according to the severity

51

of pathophysiologic condition 8.

6

2-4.

Although there are more than 1000 proteins

. In most situations, the mutant mtDNA at a low percentage

52

Previous studies have reported that the possibility of molecular therapy for

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mitochondrial diseases was to reduce the percentage of defective mtDNA through

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specific elimination of mutant mitochondrial genomes using restriction endonuclease,

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Zinc finger nucleases (ZFNs)9-11, Transcription activator-like effector nuclease



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(TALENs)12-15,

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repeats/CRISPR associated protein 9 (CRISPR/Cas9) system16-18. For example,

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Tanaka delivered a vector expressed the SmaI enzyme for cutting the point mutant

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mtDNA

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mtDNA9. Bacman and colleagues found that the percentage of normal mtDNA

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increased to 85% and 90% after treated with 14459A-mitoTALEN in two

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heteroplasmic cell lines with 10% and 45% of the wild-type mtDNA 12. Furthermore,

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the mito-TALENs successfully prevent the transmission of mitochondrial diseases by

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decreasing parent mitochondrial genomes

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introduced to edit the mtDNA in human cell 16.

19.

and

Clustered

regularly

interspaced

short

palindromic

Minczuk and colleagues used mtZFNs technology to cleave human

15.

Recently, the CRISPR/Cas9 system is

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Homologous recombination (HR) is an enzyme process conserved from

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bacteriophage to human for the repair of the double-strand break (DSB) of DNA.

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Although there were some studies on the elucidation of the DSB repair in

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mitochondria, the mechanism is still not well understood. Thyagarajan and colleagues

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found that the mitochondria protein extracts from mammalian somatic cells can

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catalyze the HR of plasmid DNA substrates in vitro

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Zouros found a high rate of mtDNA recombination in mussels and observed the

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mammalian mitochondria containing the enzymes for the HR

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that the RAD52 epistasis group proteins contributed to the HR in the mitochondria of

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Saccharomyces cerevisiae

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SWIB5 protein was associated with mitochondrial DNA in Arabidopsis thaliana and

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affected the mtDNA architecture and HR

22.

20.

In 2001, Ladoukakis and

21.

Stein et al. showed

Recently, Blomme et al. found that a mitochondrial

23.

In 2018, a work about the repair of the


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24.

78

DSB by HR in mammalian mitochondria has successfully done

All the works

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indicated that the HR is existed in mitochondria but still lacking direct evidences.

80

To date, molecular therapeutic strategies for mitochondrial diseases focused on

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mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA. The

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linearized mutant mtDNA is degradation quickly and the percentage of the wild-type

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mtDNA is elevated. However, there is no direct evidence on repairing of mutant

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mtDNA by knock-in strategy using the mito-TALENs or CRISPR/Cas9. In this study,

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we constructed a novel mito-CRISPR/Cas9 system that could edit the mtDNA,

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accompanied by the decreased of mtDNA copy number. The damaged mtDNA can be

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repaired with an exogenous single-stranded DNA (ssDNA) template via HR in human

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and zebrafish mitochondria. To our knowledge, these observations provide an in vivo

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evidence that mtDNA could be repaired through the mito-CRISPR/Cas9 system via

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HR. Thus, it proposed a possibility of gene therapy for mitochondrial disease caused

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by mtDNA mutations using mito-CRISPR/Cas9 system.

92 93

Results

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An optimized mito-CRISPR/Cas9 system for mtDNA editing in human cells and

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zebrafish.

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pSpCas9-Mito vector (Figure 1A) was constructed containing a full coding

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sequence of Cas9 (Streptococcus pyogenes) with flanking two mitochondrial targeting

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sequences (MTSs) of human and zebrafish cox8a (cytochrome c oxidase subunit 8A)

99

in this study. For the target sites, we selected two mtDNA coding genes



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NADH-ubiquinone oxidoreductase chain 1 and 4 (ND1 and ND4) of human cell and

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two sites of non-coding control region displacement loop (dloop) of zebrafish for

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targeting using mito-CRISPR/Cas9 system. ND1 and ND4 proteins are the subunits

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of NADH dehydrogenase of the electron transport chain complex I in mitochondria.

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The human mitochondrial ND1 and ND4 genes commonly carry the variations of

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mutations, which are responsible for mitochondrial diseases in Leigh's syndrome (LS)

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and Leber’s hereditary optic neuropathy (LHON)

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degeneration (AMD), mesial temporal lobe epilepsy (MTLE) and cystic fibrosis

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While dloop region is associated with the initiation of DNA replication and

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transcription

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development of cancer 32.

31,

25,

26,

age-related macular 27-30.

and the dloop sequences may play an important role in the

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The gRNA spacer sequences for targeting ND1 and ND4 were inserted into U6

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cassette of pSpCas9-Mito vector respectively, which could produce relevant gRNAs

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and mito-Cas9 protein in HEK-293T cells (Figure 1A). For zebrafish mtDNA editing

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system, gRNAs targeting the two sites of dloop were marked as dloop-1 and dloop-2,

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respectively. mito-Cas9 mRNAs of zebrafish were synthesized in vitro (Figure 1B).

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The MTSs in the N-terminus or C-terminus of Cas9 was expected to guide the Cas9

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protein to transport into the mitochondria of human and zebrafish cells.

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Mito-Cas9 protein and exogenous DNA can colocalize with mitochondrial

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marker in human cells, and gRNA exists in the isolated mitochondria

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To verify whether the mito-Cas9 protein expressed and transported into

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mitochondria, the immunofluorescence was performed with HEK-293T cells. The


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cells were transfected with the pSpCas9-Mito vector which with a 3×FLAG tag fused

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with Cas9 gene. The Figure 1C showed that the green fluorescence of

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mito-CRISPR/Cas9 protein overlapped with red fluorescence that stained by

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mitotracker Red probe. These results indicated that the mito-Cas9 protein successfully

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expressed and imported into the mitochondria with the guidance of MTS. To further evaluate whether an exogenous single-strand DNA have the capacity

127 128

to

enter

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hexachloro-fluorescein (HEX) labeled oligonucleotide (Oligo-HEX) was used to

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validate whether a single-stranded DNA can be imported into the mitochondria of

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human cell. We transfected cells with Oligo-HEX and marked the mitochondria with

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Green fluorescence. The results showed that the Oligo-HEX was localized in the

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mitochondria (Figure 1D). The similar verified conclusion can be drawn from recent

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report

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DNA can enter through the double membrane and import into the mitochondria.

18.

into

mitochondria

without

any

external

assistance.

A 5’-end

In short, these data suggest that mito-Cas9 protein and a single-stranded

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For the gRNA, we performed RT-PCR using the RNAs from pure cells and

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zebrafish mitochondria, respectively. The figure 1E is the result of agarose gel

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electrophoresis, which shows no positive band in the gel when transfection or

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microinjection without gRNA. When the gRNA was transfected and microinjected

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into cells or zebrafish, the positive band can be detected, indicating that the gRNA can

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be imported into the mitochondria of cells or zebrafish (Figure 1E).

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The mito-CRISPR/Cas9 system efficiently decrease mtDNA copy number

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Before evaluating the efficiency of the system, firstly, we test whether the PCR



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method is reliable or not. We used different treatment to test whether the ssDNA can

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be amplified as a nest-PCR template without inserting into the mtDNA. We found that

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the positive PCR band can be detected only in the intact system in both cells and

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zebrafish (Figure 2A&B), indicating that the PCR method can be used for evaluating

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the ssDNA insertion introduced by homologous recombination (HR).

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Some researchers have reported that the linearized mtDNAs were digested to 19,

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elevate the proportion of wild-type mtDNA in human cell

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embryos 15. A mitoTALENs targeted to mutated mtDNA also caused the decrease of

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mtDNA copy number in both cell and mouse

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mito-CRISPR/Cas9 system could reduce the copy number of mtDNA, transfection

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and microinjection experiments were performed using mito-CRISPR/Cas9 system

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with four gRNAs targeting sites: ND1, ND4, dloop site1 (dloop-1) and dloop site2

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(dloop-2) in HEK-293T cells and zebrafish embryos respectively. The absolute

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quantitative real-time PCR was performed to calculate the copy number of the

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mtDNA in each sample. The primers are shown in Table 2. The results showed that

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the mtDNA copy number of all samples treated by mito-CRISPR/Cas9 system

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decreased by more than 50%, compared to control, no matter with or without ssDNA

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template (Figure 2C, D), indicating our mito-CRISPR/Cas9 system successfully

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reduce the mtDNA copy number with a high efficiency.

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mtDNA mutations can caused by mito-CRISPR/Cas9 system and transmitted to

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offspring

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12, 15.

mouse oocytes and

To test whether our

To test whether the mtDNA repairing mechanisms exist in mitochondria, the


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transfection

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mito-CRISPR/Cas9 system. However, we didn’t find any indels in mitochondria by

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T7E1 assay and sanger-sequencing. We hypothesized that the NHEJ is very low

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efficient or the cleaved mtDNA degraded too quick for detecting (data not shown).

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Therefore, we performed the same procedure of transfection and microinjection

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experiments with a relevant ssDNA template to evaluate HR repairing mechanism in

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mitochondria. The ssDNA as a HR template is composed of a loxp element flanked by

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two 20 nt homologous arms. The agarose gel electrophoresis and sanger-sequencing

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results showed that the loxp element exactly inserted into the target sites of mtDNA in

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HEK-293T cells (Figure 3A, B, E) and zebrafish embryos (Figure 3C, D). All the

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PCR products of Figure 3A, B, C, and D were sequenced directly after two rounds of

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PCR amplification. For the figure 3E, the PCR products were cloned into T-vector for

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sequencing. These data suggested that the targeted mtDNA sites can be edited by HR

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mechanism using mito-CRISPR/Cas9 system.

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Further,

and

we

microinjection

verified

experiments

whether

these

were

mtDNA

performed

mutations

using

caused

this

by

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mito-CRISPR/Cas9 system can transmitted to the offspring according to maternal

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inheritance. The injected F0 female zebrafish were mated with wild-type male. Their

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zygotes were used for assay. As expected, the exogenous loxp element was detected

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in dloop-1 target site of the F1 generation in zebrafish (Figure 3F) with primer DL1-F

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and Loxp-R (Table 2). All the above indicated that the damaged mtDNA induced by

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mito-CRISPR/Cas9 system can be repaired by HR with an ssDNA template in human

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cell and zebrafish. Moreover, the mutant mtDNA can transmitted to the F1 offspring



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in zebrafish via maternal effects.

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Expression level of genes involved in NHEJ and HR pathways

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Since the mechanism of NHEJ and HR pathways in mitochondria are not clear,

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we assessed the expression profiles of genes related to nuclear genome repairing

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pathway. Real-time PCR was performed to check the mRNA expressions of major

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genes involved in nuclear NHEJ and HR pathways, as well as replication and

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transcription factors in mitochondria. KU70/KU80

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(PRKDC)

196

33

197

RAD54

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polymerase γ gene (POLG)

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important factors for mitochondrial DNA replication and transcription, respectively.

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Notably, some of nuclear pathway factors have been proved to play roles in

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mitochondria by direct or indirect evidences, such as KU80

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48,

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RPA4, RAD54 related HR pathway, and TFAM were increased, while most of the

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NHEJ related genes failed to respond except LIG4 in human cells (Figure 4A, B, C).

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We also measured the expression of homologous genes in zebrafish, and our results

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showed that the expression levels of all target genes were significantly up-regulated

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except lig4 (Figure 4D, E, F).

35,

Ligase 4 (LIG4)

33,

33, 34,

and X-ray-cross-complementation gene 4 (XRCC4)

are the major enzymes in the NHEJ pathway. RAD50 39,

RPA4

and MRE11

49

40

and BLM

41

36,

MRE11

37,

BRCA2

38,

are the vital proteins in the HR pathway. DNA

42, 43

and transcription factor A (TFAM)

45, 46,

44

are the

PRKDC 47, BRCA2

(Table 3). Our results showed that expression levels of BRCA2,

208 209

DNA-dependent kinase

Discussion


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CRISPR/Cas9 system as an emerging technique has been widely applied for 50-52.

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genome modifications in human cells, zebrafish, and others species

However,

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whether this technique can be utilized for mitochondrial DNA editing is elusive. Here,

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we reported a novel mito-CRISPR/Cas9 system that accurately knocked

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single-stranded DNA (ssDNA) into the targeting loci in the mitochondria. In this

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study, the first question is to answer whether the mito-Cas9, gRNA and ssDNA can be

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imported into mitochondria. Our results verified that mito-Cas9 and ssDNA

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(Oligo-HEX) could be colocalized with the mitochondrial marker in HEK-293T cells

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and the gRNA is imported into the mitochondria which is consistent with the recent

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report published by Loutre et al. 18. Thus, ssDNA/RNA can enter through the double

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membranes of mitochondria.

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In CRISPR/Cas9 system, Cas9/gRNA complex can recognize the specific

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sequence and cleave the target loci to generate DSB, which can be self-repaired via

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two primary mechanisms: NHEJ and HR

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have been well elucidated for genomic DNA repairing, but it is unclear whether these

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mechanisms have existed in mitochondria to date

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for mitochondrial diseases aim to cleave the mutant mtDNA and generating the

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linearized mtDNA for degradation

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was reduced by more than 50% with or without ssDNA template, indicating that the

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mito-CRISPR/Cas9 system is efficient for eliminating mtDNA. Recently, Moretton

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and Peeve demonstrated that the cleaved mtDNA is degraded quickly in vivo 43, 57. We

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speculate that the reduction of mtDNA copies is due to the DSB of mtDNA, albeit we

9-17.

53, 54.

The mechanisms of NHEJ and HR

20, 55, 56.

Most molecular therapies

Interestingly, we found mtDNA copy number



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had no direct evidences of DSB. Thus, our mito-CRISPR/Cas9 system may induce

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mtDNA DSB and the broken mtDNA is degraded quickly in mitochondria.

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Although the copy number of mtDNA is decreased by the mito-CRISPR/Cas9

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system, we found that there was no significant difference in cell viability among

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different treated groups (data not show). For zebrafish, there is no obvious phenotype

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in the early development stage. About 3 weeks after injection, the movement of

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zebrafish larvae is abnormal. Figure 4C and F showed the expression levels of two

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genes POLG and TFAM involved in mitochondrial DNA replication and transcription

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are altered in both cell and zebrafish. The reduction of the mtDNA is around 50%. We

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think that > 50% mtDNA reduction may dramatically affect the cell viability because

242

of the energy requirement. The rapid growth cells with > 50% mtDNA offset the

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dying cells with 0.99.

388

Expression levels of genes involved in NHEJ and HR

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Total RNA extraction from 30 zebrafish embryos or cells in 24-well plate using

390

RNAiso Plus reagent (Takara) according to the manufacturer’s instructions. The

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quality and concentration were determined by agarose gel electrophoresis and

392

NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, France). cDNA was

393

synthesized from 2 μg of total RNA using 5X All-In-One RT Master Mix Reagent

394

Kit (Applied Biological Materials, Richmond, BC, Canada). The real-time PCR

395

was performed using 0.2 μL of cDNA templates by SYBR Green PCR Kit (Toyobo,

396

Tokyo, Japan) on the ABI 7300 System (PerkinElmer Applied Biosystems, Foster

397

City, CA, USA) following the manufacturer’s instructions. The PCR program was

398

set with an initial denaturation step at 95 °C for 3 min, followed by 40 cycles at 95

399

°C for 20 s, 60 °C for 1 min. The dissociation curve for checking the specificity of

400

PCR production was acquired by adding a step of 95 °C for 15 s. The expression

401

level of target genes was calculated by 2−ΔΔCt method.

402

Statistical analysis

403

For statistical analysis, one-way analysis of variance (ANOVA) was applied to



404

calculate the differences between the control and experimental groups by GraphPad

405

Prism version 5.01 (GraphPad Software, USA). A value of p

Knock-In Strategy for Editing Human and Zebrafish Mitochondrial DNA

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