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. 2024 Nov 20;13(12):3926–3935. doi: 10.1021/acssynbio.4c00265

CRISPR Diagnostics for Quantification and Rapid Diagnosis of Myotonic Dystrophy Type 1 Repeat Expansion Disorders

Koji Asano †, Kazuto Yoshimi †,‡,*, Kohei Takeshita §, Satomi Mitsuhashi ∥, Yuta Kochi ⊥, Rika Hirano †, Zong Tingyu †, Saeko Ishida †, Tomoji Mashimo †,‡,*
PMCID: PMC11669157  PMID: 39565688

Abstract

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Repeat expansion disorders, exemplified by myotonic dystrophy type 1 (DM1), present challenges in diagnostic quantification because of the variability and complexity of repeat lengths. Traditional diagnostic methods, including PCR and Southern blotting, exhibit limitations in sensitivity and specificity, necessitating the development of innovative approaches for precise and rapid diagnosis. Here, we introduce a CRISPR-based diagnostic method, REPLICA (repeat-primed locating of inherited disease by Cas3), for the quantification and rapid diagnosis of DM1. This method, using in vitro-assembled CRISPR-Cas3, demonstrates superior sensitivity and specificity in quantifying CTG repeat expansion lengths, correlated with disease severity. We also validate the robustness and accuracy of CRISPR diagnostics in quantitatively diagnosing DM1 using patient genomes. Furthermore, we optimize a REPLICA-based assay for point-of-care-testing using lateral flow test strips, facilitating rapid screening and detection. In summary, REPLICA-based CRISPR diagnostics offer precise and rapid detection of repeat expansion disorders, promising personalized treatment strategies.

Keywords: CRISPR-Cas3, triplet repeat diseases, CRISPR diagnostics, quantitative detection, Myotonic dystrophy type 1, genetic diagnosis

Introduction

Early genetic profiling related to clinical symptoms is crucial in various diseases, playing a significant role in patient health management and treatment selection. Genetic diagnostics has become a primary tool since advances such as microarrays and next-generation sequencing (NGS), allowing simultaneous detection of multifactorial mutations, for example gene panel testing.1,2 However, regions with complex structures remain challenging for NGS sequencing analysis, such as gene duplication regions, repeat sequences, chromosomal structural variations, and GC-rich regions, and so these are still detected using classical techniques such as Southern blotting and PCR.3−6

Myotonic dystrophy type 1 (DM1) serves as a challenging example for genetic diagnostics. DM1, a neuromuscular disorder, is attributed to abnormal expansion of CTG triplet repeats within the DMPK gene. The severity of the neuromuscular dystrophy-like symptoms increases proportionally with the number of CTG repeats, with severe cases known to have 1000–2000 repeats, or more.7 Current diagnostic methods for CTG repeat expansion in clinical settings primarily use repeat-primed PCR (RP-PCR) and Southern blotting. While PCR combined with capillary electrophoresis enables convenient quantification of repeat length, it has low sensitivity for detecting repeats of more than 150 copies, compromising the reliability of repeat quantification.8 Conversely, Southern blotting can detect long repeats, but requires substantial DNA samples and involves longer turnaround time and higher cost than PCR. Thus, genetic diagnosis of triplet-repeat diseases, such as DM1, requires a nucleic acid detection method that is both convenient and able to accurately quantify repeat numbers.

CRISPR-based diagnostic (CRISPR-Dx) technology, a simple and rapid nucleic acid detection method utilizing CRISPR and Cas DNA-recognition enzymes, has been employed widely in recent years, primarily targeting viruses.9−12 This includes SHERLOCK (specific high-sensitive enzymatic reporter unlocking), relying on Cas1313, and DETECTR (DNA endonuclease-targeted CRISPR trans reporter), relying on Cas12a.14 By further combining these methods with lateral flow test strips, the cleavage of ssDNA probes by the CRISPR system can be easily and quickly visualized as bands, and this approach has been applied to the detection of novel coronaviruses and influenza viruses.15−17 Additionally, we have developed CONAN (Cas3-operated nucleic acid detection), using type I CRISPR-Cas3 from the class 1 CRISPR system, which exhibits non specific collateral single-stranded DNA cleavage activity following specific binding to cis-target DNA via programmable CRISPR RNA (crRNA).17 CRISPR-Cas12 and CRISPR-Cas13 can cleave the target with a single factor, whereas CRISPR-Cas3 works by forming a complex called Cascade with multiple factors, including Cas5, Cas6, Cas7, Cas8, and Cas11, along with crRNA. The Cascade complex is known to recognize and bind to a 32-base pair sequence, which is longer than the 20-base pair recognition sequence of Cas9, and is therefore believed to recognize targets with greater specificity. In addition, unlike Cas12a, the Cas3 system can recognize different protospacer adjacent motif (PAM) sequences and has high single-base specificity, making it promising for viral and genetic CRISPR-Dx. Notably, while Cas12 recognizes TTT as a PAM sequence and cannot directly recognize CAG/CTG repeats, Cas3 can recognize CTG or CAG as a PAM sequence, suggesting its potential utility in repeat sequence detection.17,18

Here, we report the establishment of a system for rapid and quantitative CTG repeat detection using the CRISPR-Cas3 protein complex. This method, named REPLICA (repeat primed locating of inherited disease by Cas3), offers rapid quantification of patient genomic DNA, enabling classification of disease severity. Furthermore, when combined with isothermal amplification and paper-based tests, it also provides convenient and rapid detection of CTG repeats, promising potential point-of-care testing (POCT) applicability.

Results and Discussion

CTG Repeat Quantification Assay Using In Vitro-Assembled CRISPR-Cas3

Based on previous reports narrowing the candidate CRISPR-Cas3 PAM sequences to CAG or CTG,18 we designed two crRNAs and synthesized the Cascade protein complex to evaluate cleavage activity. A crRNA containing a 32-base sequence complementary to CAG or CTG repeats was designed and the Cascade for detection of CAG or CTG repeats was synthesized using conventional Escherichia coli (E. coli) purification methods (Figure S1A). While the protein complexes demonstrated in vitro collateral cleavage activity, they showed significant batch-to-batch variation and poor signal-to-noise ratio (Figure S1B,C). Gel electrophoresis of crRNAs extracted from the E. coli-synthesized Cascade complex revealed smeared bands, suggesting incomplete Cascade formation because of the RNA repeat complexity (Figure S1D). Therefore, we attempted to address this issue by synthesizing each Cas factor using Sf9 insect cells, which have been effective in synthesizing functional Cas3 proteins,18 and His-tag extraction, followed by in vitro complex formation with synthesized crRNAs (Figure 1A). All tagged Cascade factors were expressed in Sf9 cells using a P2A sequence and purified via nickel resin columns and gel filtration chromatography, successfully yielding a purified solution containing all factors (Figures 1B and S2). To investigate collateral cleavage activity when mixing them with crRNA precursors in vitro, we combined existing crRNAs targeting the mouse Tyr gene and conducted in vitro assays for collateral cleavage activity. Comparison of the activity of the recombinant E. coli Cascade and the in vitro-assembled Cascade from Sf9 cells revealed comparable cleavage activity at the same target DNA concentration (Figure S3). Evaluation of cleavage activity using in vitro-assembled Cascade complexes created by mixing crRNAs targeting CTG and CAG repeat sequences revealed no cleavage activity without a target sequence, whereas significant signals were observed in the presence of target sequences (Figure 1C,D), increasing in a concentration-dependent manner (Figure S4). Notably, detection of CAG repeats yielded more pronounced signals, consistent with the known preference for CAG over CTG as a PAM sequence. These results demonstrate the successful synthesis of a CRISPR-Cas3 protein complex capable of evaluating CTG repeat expansion.

Figure 1.

Figure 1

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In vitro collateral cleavage activity of CRISPR-Cas3 for CTG repeats. (A) Schematic representation of the in vitro assembly of the Cascade-crRNA complex. pre-crRNA was added to a mixture of Cascade components containing Cas5, Cas6, Cas7, Cas8, and Cas11 proteins, and incubated at 37 °C for 10 min. (B) Each of Cas5, Cas6, Cas7, Cas8, and Cas11 were expressed using the baculovirus expression system in Sf9 insect cells. M: protein marker. (C) Time courses of collateral ssDNA cleavage activity measured by incubation of E. coli Cas3 and Cascade with or without a 60-bp dsDNA activator containing a target sequence containing CTG (CAG) repeats and an FQ-labeled ssDNA probe in reaction buffer containing MgCl2, CoCl2, and ATP for 30 min at 37 °C. (D) CRISPR-Cas3-mediated collateral ssDNA cleavage activity at 10 min after targeting CTG (CAG) repeat-dsDNA in fragments, quantitatively represented by relative fluorescence units (RFU). Means (n = 3), and standard deviations. ***p < 0.001, one-way ANOVA with posthoc test.

REPLICA: CRISPR-Dx for Quantitative Detection of CTG Repeats in DM1 Patients

CTG repeat expansion length is related to the severity of triplet-repeat diseases such as DM1. Quantifying the CTG repeat expansion length with CRISPR-Cas3 requires direct recognition of the repeat sequence (Figure 2A). To evaluate the quantitative assessment of CTG repeat expansion in the DMPK gene, we prepared multiple plasmids containing the DMPK CTG repeat sequence, sourced from Addgene. Subsequently, we investigated whether the obtained signal intensity corresponded to each sequence count. Our findings indicated that, while minimal signal was detected from low copy numbers typically found in unaffected individuals, significantly heightened signals were observed when over 200 copies were present in patients. Furthermore, we noted a repeat-count-dependent increase in signal intensity (Figure 2B). These outcomes suggest the potential of the CTG-repeat optimized CRISPR-Cas3 system for facilitating convenient CTG repeat measurement in DM1 patients.

Figure 2.

Figure 2

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Quantitative detection of CTG repeats by CRISPR-Cas3. (A) Illustration showing the concept of the detection of CTG repeat expansion by CRISPR-Cas3. More Cascade complexes bind DNA from DM1 patients with CTG repeat expansion, resulting in a higher intensity ssDNA cleavage signal. Conversely, in unaffected individuals with less than 35 CTG repeats, only a very low cleavage signal is obtained. (B) Quantitative assay detecting plasmids containing exons 11 to 15 of the DMPK gene and CTG repeats of 0, 12 (unaffected), 240, 480, or 960 (patient-like) (left). ssDNA cleavage signals are represented by RFU per min; graph shows increasing rate of RFU/min (right). Means (n = 3), and standard deviations. *p < 0.05, **p < 0.01, one-way ANOVA with posthoc test.

Next, we aimed to diagnose DM1 patients with high probability by specifically and accurately determining the number of CTG repeats in the DMPK gene using this CRISPR-Cas3 system (Figure 3A). We obtained genomes from DM1 patients and unaffected individuals from the Coriell Institute (https://www.coriell.org/1/NIGMS), with predicted repeat numbers based on Southern blotting. Additionally, to enhance the accuracy of repeat numbers, we extracted and analyzed data from the literature where the repeat numbers of these samples were verified using Southern blotting at multiple institutions.19 Amplifying long repeat regions from genomic DNA is challenging with conventional PCR, hence we used repeat-primed PCR (RP-PCR) after confirmation of amplification (Figure S5). To mitigate the impact of dNTPs and primers in the RP-PCR products on the collateral cleavage activity of CRISPR-Cas3, we examined dilutions of the RP-PCR products. We found that a 2.5-fold dilution of the PCR products yielded the highest signal from patient samples (Figure S6). Moreover, we investigated the length and concentration of the tailed repeat-primed (RP) reverse primers used in RP-PCR to anneal to the CTG repeats. We determined that, compared with other primers, those with four CAG repeats showed reduced amplification of repeats in unaffected individuals and specific detection of repeat expansions (Figure S7). Additionally, when optimizing the concentration of the RP primers, we found that using 40 and 100 nM improved the signal-to-noise ratio (Figure S8). Screening various PCR enzymes demonstrated that Tks Gflex or KOD One DNA polymerases gave specific amplification of CTG repeats based on their length (Figure S9). Furthermore, we tested the addition of 5 mM ATP to enhance helicase activity, resulting in higher signals from long CTG repeat-derived amplicons (Figure S10). By enhancing the ATP-dependent helicase activity of Cas3, we were able to unwind long CTG repeats that form complex secondary structures, thereby establishing conditions that yield higher collateral cleavage activity from longer CTG repeats. Under these optimized conditions, we achieved reliable detection of DM1-positive patients (Figure 3B). We also found a strong correlation between fluorescence values and expected repeat numbers within the clinically important range of up to 1000 repeats (y = 1.40x, R2 = 0.93, Figure 3C). For CTG repeat lengths greater than 1000, we noticed that the REPLICA signal did not increase as much as expected, and the method became less sensitive in quantifying these very long repeats. Despite this limitation, when we analyzed all our data, including these longer repeats, we still found a very strong relationship between the REPLICA signal and the repeat length using a logarithmic scale (y = 266.12 ln(x)–753.38, R2 = 0.90, Figure S11). This result indicated that our method remains highly reliable even for extremely long CTG repeats, albeit with reduced sensitivity.

Figure 3.

Figure 3

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Quantitative detection of CTG repeats in DM1 patients using the REPLICA method. (A) Illustration showing the concept of the REPLICA assay. Quantitative detection of repeats is achieved by detecting amplified products containing various lengths of CTG repeats from the CTG repeat region of the DMPK gene locus using REPLICA, obtained by triplet repeat-primed PCR (RP-PCR). (B) REPLICA assay from genomic DNA of DM1 patients with various CTG repeats or from genomic DNA of unaffected individuals. (C) Correlation between cleavage signal in the REPLICA assay and CTG repeat number up to 1000 repeats. (D) Cleavage signal in the REPLICA assay for Coriell samples classified by symptoms (healthy, mild, classical, or congenital) based on available clinical information. (E) REPLICA assay for detecting clinical samples from three DM1 patients or 30 non-DM1 individuals. ssDNA cleavage signals are represented by RFU per min; graph shows increasing rate of RFU/min (right). Means (n = 3), and standard deviations. *p < 0.05, ***p < 0.001, one-way ANOVA with posthoc test.

To validate the repeat lengths of the clinical samples used in this study, we quantified and verified the repeat numbers in representative 11 DM1 patient samples using long-read sequencing technology with Nanopore. The signal values obtained by the REPLICA method showed a high correlation with the results from long-read sequencing and were comparable to those obtained by Southern blot analysis (y = 1.20x, R2 = 0.84, vs y = 1.24x, R2 = 0.93), confirming that our method maintains quantitative accuracy (Figure S12).

Furthermore, using the clinical onset symptoms shown in the Coriell database, we classified the samples into three groups: mild (onset age, 31-70; CTG repeat size, 50-150), classical (onset age, 11-30; CTG repeat size, 150-1000), and congenital (symptoms characteristic of congenital conditions, such as significant facial diplegia or severe generalized weakness; onset age, 0-10 or NA; CTG repeat size >1000). As a result, we observed a significant increase in the cleavage signal in a severity-dependent manner (Figure 3D). These results demonstrate reliable and repeat-size-dependent detection of DM1-positive patients, enabling diagnosis and severity assessment of DM1 patients.

To validate the precision of our method, 33 genomic samples, including three DM1 patients diagnosed by Southern blotting and 30 non-DM1 patients collected at St. Marianna University School of Medicine in Japan, were subjected to CRISPR-Cas3 diagnosis in triplicate. Results revealed no signals in all 30 non-DM1 patient-derived genomes, while strong fluorescent signals were obtained in the three DM1 patient-derived samples, with 100% positive predictive agreement and 100% negative predictive agreement (Figure 3E). Predicted repeat numbers of the three genomes from the DM1 patients from the relative fluorescence unit (RFU) values were approximately 949, 248, and 524, respectively, consistent with the estimation in the clinic of 500–1000 repeats by Southern blotting, not only suggesting the presence of repeat disease but also providing an estimation of severity. To validate the absence of CTG repeat expansion in non-DM1 samples, the repeat number was quantified using Sanger sequencing. As a result, it was confirmed that none of the 33 control samples had abnormal repeat expansions exceeding 35 repeats (Table S3). Integrating the results from all 59 clinical samples from Coriell institute and St. Marianna University achieved 100% specificity in detecting CTG repeat expansion. These findings demonstrate the ability to quantitatively evaluate CTG repeat numbers in DM1 patient genomes using our optimized method, which we named REPLICA (repeat primed locating of inherited disease by Cas3). Quantifying CTG repeat length in repeat disorders by REPLICA allows definitive molecular diagnosis by discriminating affected from unaffected individuals, while simultaneously enabling prediction of severity among patients to guide tailored therapeutic strategies.

REPLICA-Based Assay for Rapid Detection of CTG Expansion

The diagnosis of triplet-repeat diseases in clinical practice, as for the diagnosis of infectious diseases, requires POCT that does not require fluorescence measurement equipment such as a thermal cycler or plate readers. The simple and early diagnostic method would diminish diagnostic barriers for patients and high-risk families, enabling identification of undetected carriers by conventional methods. Identification and stratification of patients by repeat number would facilitate appropriate genetic counseling, management of reproductive risks, and timely treatment. To enable POCT of DM1 patients, we optimized the REPLICA method using lateral flow test strips (Figure 4A). Convenient diagnostic methods using test strips primarily use one-step isothermal PCR incubation.20 Recombinase polymerase amplification (RPA) generally uses longer primers than PCR primers. Therefore, CTG repeat regions annealing to reverse primers with five, six, and seven repeats were examined. The result showed that five repeats gave a signal corresponding to the number of CTG repeats in the genome (Figure S13). When testing primer concentrations of 100, 300, and 500 nM, the amplification was proportional to the repeat length at a 5× diluted forward primer concentration of 100 nM, with a significant signal increase observed even at 70 repeats (in a mosaic with 56 repeats) (Figure S14). Furthermore, while the recommended RPA reaction temperature is 37–42 °C, previous reports have suggested that lower temperatures are suitable for amplification of GC-rich repeat sequences.21 Therefore, RPA reaction temperatures of 32, 37, and 42 °C were investigated, showing that 37 °C gave the highest cleavage signal (Figure S15). After these optimizations, detection on test strips was performed using gold nanoparticle-conjugated anti-FITC antibodies as reporter probes, generating a signal on the second line (positive) within 5 min of reporter cleavage.17 Using RPA-REPLICA in a one-pot assay, we detected CTG repeats of approximately 1000 copies within 1 h (Figure 4B). To demonstrate suitability for rapid DM1 diagnosis, we examined DM1 patient-derived genomes using this system. Clear signal bands were obtained in 22 of 23 patient genomes (excluding a carrier with somatic mosaicism of 70 repeats), successfully detecting repeat expansion on the test strip (Figures 4C,D and S16). Three unaffected donor genomes showed no clear bands at the test band site, giving a negative result. Furthermore, fitting the fluorescence values from these samples and expected repeat numbers obtained from Southern blotting to a logarithmic approximation curve yielded an R2 value of 0.69, indicating a moderate correlation (Figure 4E,F). These results demonstrate the potential for DM1 diagnosis by POCT using test strips and REPLICA in combination.

Figure 4.

Figure 4

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RPA-REPLICA for point of care testing (POCT) for DM1. (A) Schematic diagram of RPA-REPLICA for POCT. Amplification of various lengths of CTG repeat regions at isothermal conditions of 37 °C using RPA, followed by an ssDNA cleavage assay with REPLICA, and detection of a positive band indicating repeat expansion in approximately 5 min using a test strip. (B) Detection of repeats using a test strip. The red arrow indicates the positive band for CTG repeats. T, test band; C, control band. (C,D) Detection of CTG repeats in RPA-REPLICA for POCT. NC, negative control; HC, healthy control. (E) Detection of amplified CTG repeats with RPA-REPLICA. (F) Comparison between CTG repeat lengths and ssDNA cleavage signals.

Conclusions

In this study, our innovative CRISPR-Dx approach using a reconstructed Cas3 system represents a significant advance for the rapid and accurate diagnosis of DM1 and other repeat disorders. In the REPLICA method, by optimizing the RP-PCR conditions, it was possible to quantify even very long repeats. This highly stable amplification method prevented any false negatives due to amplification failures in all 33 samples. This method surpasses traditional techniques like PCR and Southern blotting, offering high sensitivity and specificity. Its potential extends beyond DM1 to the diagnosis of other repeat expansion diseases. The development of this technique enables efficient and accessible screening to identify the patients with undiagnosed triplet repeat diseases, with prospects for blood sample detection and automation, potentially revolutionizing genetic disease screening. There remains the possibility that the variability in signals obtained using the REPLICA method is due to DNA amplification. Therefore, further improvement of the amplification conditions could potentially result in a more accurate method. While challenges remain in enhancing accuracy and efficiency, and further validation studies are required to establish clinical utility and reliability, CRISPR-based diagnostics hold immense promise for both research and clinical applications.10,22−24 The ability to detect repeat expansions with high precision, even in crude samples, without extensive sample preparation, is a significant advantage over existing methods. While enough genomic DNA can be obtained from the blood of DM1 patients, future improvements to enable the detection of repeat expansions from smaller amounts of DNA present in urine and other sources could make this method more applicable in clinical settings.

The findings of this study, showing a consistent correlation between repeat length and signal strength, reinforce the accuracy of the technique, validated by previous research methods including Southern blot analyses and sequencing. In recent years, it has been reported that CTG/CAG repeat sequences can be detected using long-read sequencing technology, allowing for the accurate quantification of the length of these repeat sequences.25,26 However, due to the difficulty in amplifying CTG/CAG repeats, enrichment steps using methods such as Cas9 enrichment are required. Additionally, the need for expensive Flow cells and specialized software and knowledge for analysis suggests that the application of these methods to diagnostics will take time. In contrast, the REPLICA method allows for the simple and rapid quantification of CTG repeat lengths over a wide range, making it possible to timely provide classifications such as healthy, mild, classical, and congenital. In the REPLICA method, even when using the same amount of genomic DNA, it is not possible to accurately quantify the number of repeats due to heterogeneity in somatic cells. Therefore, it is believed that the complementary use of the REPLICA method and long-read sequencing technology could contribute to more accurate diagnosis of repeat expansion diseases. Although a cure for DM1 is currently unavailable, ongoing clinical trials exploring treatments including antisense oligonucleotides (ASOs),27 erythromycin,28 and CRISPR-based genetic therapies29 offer promising avenues for future therapeutic options and symptom management. When these treatment methods including a gene therapy targeting repeat expansion will be developed, early detection would not only increase the possibility of treatment but even lead to prevention of disease onset. Thus, the widespread adoption of CRISPR-Dx has the potential to estimate disease prevalence, as well as to assist in distinguishing DM1 from other conditions, and to inform future treatment strategies tailored to individual patients.

Methods

Expression and Purification of Cas3, the Mixture of Cascade Components, and Recombinant Cascade

We employed a method to express recombinant E. coli Cas3 (EcoCas3) using a baculovirus expression system, as described previously.17,18 Briefly, we cloned EcoCas3 cDNA with an octa-histidine tag and a six asparagine–histidine repeat tag into a pFastbac-1 plasmid (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The TEV protease recognition site was also inserted between the tags and EcoCas3 to enable tag removal.

To produce the mixture of Cascade components, a plasmid was designed in which each Cascade component gene was cloned into pFastbac-1 by incorporating nuclear localization signal (NLS) sequences at the N- and C-termini and a His-tag sequence at either the N- or C-terminal, linked by 2A-peptide. Expression and purification of the mixture of Cascade components in Sf9 was performed by the Bac-to-Bac baculovirus expression system using Sf9 insect cells (Thermo Fisher Scientific Inc., Carlsbad, CA, USA). Then, the plasmid containing the Cascade genes in tandem with 2A-peptide was transformed into DH10Bac competent cells. Each purified bacmid was transfected into Sf9 cells, and high-titer baculoviruses were acquired by repeatedly infecting new cells with baculovirus in the culture supernatant. The acquired baculovirus was added to Sf9 culture cells at two multiplicities of infection (MOI), then cultured at 28 °C for 24 h. After the infection, the culture temperature was lowered to 20 °C for 3 days for protein expression. Sf9 cells were collected and stored at −80 °C until purification. After ultrasonic disruption of cells, the homogenized sample was ultracentrifuged and the supernatant, including the Cas proteins, was collected. The Cas proteins were purified using nickel affinity resin (Ni-NTA, Qiagen). Superdex 200 Increase 10/300 GL (Cytiva, Tokyo, Japan) was used for gel filtration chromatography.

The recombinant Cascade optimized for CTG repeats was purified in accordance with previously reported methods.18,30,31 Briefly, the recombinant Cascade ribonucleoproteins (RNPs) were expressed in JM109 (DE3) by cotransformation with three pET plasmids series which are compatible with each other: one encoding a hexahistidine tag and an HRV 3C protease recognition site in the N-terminus of Cas11 (plasmid pCDFDuet-1); one containing the genes encoding Cas5, Cas6, Cas7, Cas8, and Cas11 proteins (plasmid pRSFDuet-1); and one encoding the crRNA (pACYCDuet-1). We prepared each plasmid at a concentration of 500 ng/μL and mix them in equal molar ratios before transforming them into the E. coli host strain. The transformed bacteria were cultured in 2XYT medium at 37 °C with 130 rpm. After the OD600 became 0.6 to 0.8, IPTG was added (final concentration, 0.4 mM) and the cells were cultured at 26 °C with 110 rpm for 16 h. The expressed Cascade-crRNA RNPs were purified by Ni-NTA resin. After removing the hexahistidine tag using HRV 3C protease, the recombinant Cascade RNPs were further purified by size-exclusion chromatography in 350 mM NaCl, 1 mM DTT, and 20 mM HEPES-Na (pH 7.0), and size-evaluated by SDS-PAGE. All plasmid sequences we used are uploaded as GenBank files.

Patient DNA Samples and Study Protocol Approvals

We obtained genomes from DM1 patients and unaffected individuals from the Coriell Institute (https://www.coriell.org/1/NIGMS) which are listed in Table S2. We also utilized samples from three patients with DM1 and 30 disease controls, comprising 15 patients with genetically and clinically defined repeat expansion diseases, and 15 patients with cerebrovascular diseases. DNA samples were extracted from peripheral blood using the QIAamp DNA Blood Kit (Qiagen, Venlo, Netherlands). Written informed consent and disclosure were obtained from the patients. The study protocol received approval from the Institutional review board of St. Marianna University School of Medicine (#4983).

DNA and RNA Preparation

For the activator templates, 60 bp oligonucleotide fragments of CTG (CAG) repeats were purchased from Eurofins Genomics (Tokyo, Japan). The plasmids (pBItetDMPKSGFP, 0 repeat, Cat#96903; pBItetDT12nGFP, 12 repeats, Cat#80420; pBItetDT240GFP, 240 repeats, Cat#96904; pBItetDT480GFP, 480 repeats, Cat#96905; pBItetDT960GFP, 960 repeats, Cat#80419) were purchased from Addgene (Watertown, MA, USA). These plasmids have tcgag sequences interrupting every 60 bases of CTG repeats. The primers used for RP-PCR and RPA were from P. Dryland et al.,32 or modified using Primer3. All primer sequences are listed in Table S1.

Pre-crRNAs (including repeat-spacer-repeat sequences) for Cascade formation were generated by the in vitro transcription (IVT) method using the MEGAshortscript T7 Transcription Kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. DNA fragment templates containing a T7 promoter, repeats, and a single spacer were used for IVT. The pre-crRNAs were then purified using the Monarch RNA Cleanup Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocol.

Repeat-Primed PCR (RP-PCR)

Each 25 μL PCR reaction comprised KOD One PCR Master Mix (TOYOBO, Osaka, Japan), 200 nM forward primer, 100 nM reverse primer, 200 nM tail primer, 0.5 M Betaine (Wako), and 50 ng of genomic DNA, except when optimizing the PCR conditions. The PCR amplification conditions comprised an initial denaturation at 95 °C for 5 min then 30 cycles of denaturation at 98 °C for 45 s, annealing at 72 °C for 30 s with extension at 82 °C for 3.5 min, and a final extension at 72 °C for 10 min. The PCR products were subjected to REPLICA assay.

The Sequencing of CTG Repeat Sequences

To quantify the number of CTG repeats in non-DM1 samples, the CTG repeats at the DMPK gene locus were amplified by PCR. The PCR amplification conditions comprised an initial denaturation at 94 °C for 1 min then 40 cycles of denaturation at 98 °C for 45 s, annealing at 66 °C for 30 s with extension at 82 °C for 3.5 min, and a final extension at 72 °C for 10 min. Sanger sequencing was performed by Azenta Life Sciences (Tokyo, Japan) using DMPK-Seq primers.

Long-Read Sequencing of CTG Repeat Expansion

Long-read sequencing to determine the number of CTG repeat expansion in several DM1 patient samples was performed according to the method as previously described,33 and the number of CTG repeats was determined. Briefly, library preparation for CTG repeat was performed using the Cas9 enrichment kit (SQK-CS9109, Oxford Nanopore Technologies, Oxford, U.K.) according to the manufacturer’s protocol. Nanopore long-read sequencing was performed on a PromethION flow cell (PRO002, Oxford Nanopore Technologies, Oxford, U.K.) using the P2 solo sequencing platform. Data analysis was done as previously described using LAST and tandem-genotypes.33−35 Repeat copy number changes relative to the reference genome for 2 alleles were predicted using the tandem-genotypes -o option.

Recombinase Polymerase Amplification

To detect DNAs, isothermal amplification and RPA were performed using the TwistAmp Basic kit (TwistDx, Maidenhead, UK) according to the manufacturer’s protocol. Template DNAs were amplified by incubation at 37 °C for 40 min. Excluding the optimization of the RPA conditions, 500 nM forward primer, 100 nM reverse primer, and 75 ng of genomic DNA were used.

In Vitro Collateral Cleavage Assay

REPLICA assays based on the collateral cleavage activity of CRISPR-Cas3 were performed as previously described17 with modifications. Cascade-crRNA complex equivalent to 100 nM was prepared by mixing equal volumes of 250 ng/mL pre-crRNA and 0.8 mg/mL of the mixture of Cascade components, followed by incubation at 37 °C for 10 min. Then, DNA templates equivalent to 100 nM were added to the Cascade-crRNA complex, with 400 nM Cas3 and 5 mM ATP in CRISPR-Cas3 system working buffer (60 mM KCl, 10 mM MgCl2, 10 μM CoCl2, and 5 mM HEPES-KOH, pH 7.5). The ssDNA reporter probe (5′-/5HEX/AAGGTCGGA/ZEN/GTCAACGGATTTGGTC/3IBFQ/-3′) (100 nM) was added, and the probe’s cleavage-related change in the fluorescent signal was measured every 30 s for up to 60 min with incubation at 37 °C.

Lateral Flow Assay

For lateral flow readouts, CRISPR-Cas3 system reaction mixture was added to the Cascade-crRNA complex at the equivalent of 100 nM, with 200 nM Cas3 and 5 mM ATP in the working buffer. A 2 μL aliquot of the amplicon was added to 18 μL of Cas3, the Cascade-crRNA complex, and 50 nM ssDNA reporter probe (5′-/5-FITC/TAGCATGTCA/3-Biotin/-3′). The mixture was incubated for 30 min at 37 °C. After adding 80 μL dilution buffer, a lateral flow strip provided from TAUNS Laboratories Inc. (Shizuoka, Japan) was added to the reaction tube and the result was visualized after approximately 5 min. A lower band close to the sample pad indicated a negative result (uncut probes), whereas an upper band close to the top of the strip indicated the 5′ end of the cut probes. Emergence of an upper band indicated a positive result. The test band intensities on the lateral flow strips were quantified by the gray values using the ImageJ tool and were visualized on a heatmap (Figure 4C).

Acknowledgments

We thank Ms. Hiromi Taniguchi and Ms. Yuko Yamauchi at the University of Tokyo for their technical assistance with in vitro assays, Ms. Sachiko Yamamoto and Ms. Shuku Saji at the RIKEN SPring-8 Center for their technical assistance with protein extraction and purification, and Ms. Eri Nonoyama at St. Marianna University School of Medicine for their technical assistance with Nanopore sequencing. We are also grateful to Catherine Perfect, MA (Cantab), from Edanz (https://jp.edanz.com/ac), for editing a draft of this manuscript. This research was made possible through the Allen Cell Collection, available from Coriell Institute for Medical Research. This study was supported in part by JSPS KAKENHI from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) (19KK0401, 22K19238, 22H02266, 23H00367) and a grant from the Japan Agency for Medical Research and Development (23ck0106807, 24bm12230009, 223fa627001).

Glossary

Abbreviations

DM1

Myotonic dystrophy type 1

CRISPR

clustered regularly interspaced short palindromic repeat

Cas

CRISPR associated protein

REPLICA

repeat-primed locating of inherited disease by Cas3

RPA

Recombinase Polymerase Amplification

ssDNA

single-stranded DNA

FQ

fluorophore quencher

PAM

protospacer adjacent motif

crRNA

CRISPR RNA

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.4c00265.

  • Characterization of recombinant Cascade; purification of mixture of Cascade components; validation of the in vitro-assembled Cascade; electrophoresis of RP-PCR; optimization of RP-PCR conditions; optimization of REPLICA assay; correlation between cleavage signal and CTG repeat number in the REPLICA assay for DM1 samples; validation of CTG repeat length in DM1 samples by long-read sequencing and comparison with Southern blot and REPLICA assay; optimization of the RPA conditions; REPLICA for POCT lateral flow assay results; the oligonucleotide sequences used in this study; clinical samples obtained from the Coriell Institute; number of CTG repeats of non-DM1 samples (PDF)

  • All plasmid sequences we used are uploaded as GenBank files (ZIP)

Author Contributions

KY and TM conceived the study, analyzed the data, and wrote the paper. KA performed most of the experiments and analyzed the data with assistance from RH and KY. KT performed the preparation and purification of all CRISPR-Cas proteins. SM, ZT and SI prepared the clinical samples for CRISPR diagnostic testing. SM and YK performed long read sequencing and analyzed the data. All authors have read and approved the manuscript before submission.

The authors declare no competing financial interest.

Special Issue

Published as part of ACS Synthetic Biology special issue“Diagnostics”.

Supplementary Material

sb4c00265_si_001.pdf (1.4MB, pdf)
sb4c00265_si_002.zip (52.9KB, zip)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

sb4c00265_si_001.pdf (1.4MB, pdf)
sb4c00265_si_002.zip (52.9KB, zip)

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