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CRISPR-Clear: a Fieldable Detection Procedure for Potential CRISPR-Cas9 Gene Drive Based Bioweapons

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CRISPR-Clear: a Fieldable Detection Procedure for Potential CRISPR-Cas9 Gene Drive Based Bioweapons CRISPR-Clear: a fieldable detection procedure for potential CRISPR-Cas9 gene drive based bioweapons Anna C. Nieuwenweg,1,2 Martijn M. van Galen,1 Angelina Horsting,1 Jorrit W. Hegge,2 Aldrik H. Velders,1 Vittorio Saggiomo1* 1 Laboratory of BioNanoTechnology, Wageningen University and Research, Axis, Bornse Weilanden 9, 6708 WG Wageningen, the Netherlands. E-mail: [email protected] 2 Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6708WE Wageningen, The Netherlands Abstract: Rapid progression in genetic modification research has made gene editing increasingly cheaper and easier to use. CRISPR-Cas9 for example, allows for the specific alteration of the genome of an organism with relative simplicity and low costs. This raised a worrying question; can genetic modification techniques be used to create novel bioweapons? A specific scenario is the initiation of a synthetic gene drive for malicious purposes. A synthetic gene drive can be used to quickly spread a mutation through an entire population. This mutation could alter vectors in such a way that they will spread human diseases or eradicate essential organisms. Since a gene drive spreads efficiently through a population, timely detection is essential. Thus, a quick and field deployable screening method is needed to counteract the malicious use of gene drives. Here, we show a battery-operated, sensitive screening method, named CRISPR-Clear, for the detection of gene drive modified organisms. CRISPR-Clear is based on the combination of three components: 1) A DNA amplification technique known as loop-mediated isothermal amplification (LAMP) for detecting the presence of a gene drive; b) a portable battery-operated Arduino device which heats up the sample to allow DNA amplification, and c) a naked-eye visualization of the results. We designed and tested six LAMP primers targeting a Cas9 endonuclease-based gene drive, assembled a battery-operated Arduino device and tested the naked-eye visualization method. In addition, we were able to detect the presence of the Cas9 gene, extracted from a transformed bacteria, providing a proof-of-concept of the CRISPR-Clear device. Introduction: The rapid progression in genetic modification research has made specific genome editing increasingly cheaper and easier to use [1]. Leading in the current genome editing techniques is the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 or CRISPR-Cas9 [2]. CRISPR-Cas9 was first discovered in a natural immune system that protects bacteria against foreign DNA. This natural system can be converted into a potent genome editing tool, capable of making specific modifications without leaving any traces in the modified DNA. CRISPR are palindromic sequences on the bacterial genome and are part of the bacterial defense systems against alien DNA. The repeats contain DNA sequences from earlier encounters. This library of foreign DNAs is used to detect and destroy viruses or plasmids that bear that specific DNA. With the use of RNA harboring these sequences, CRISPR associated (Cas) proteins recognize and cut the alien DNA when the bacterial cell is invaded. Several CRISPR-Cas systems, among which CRISPR-Cas9 have been repurposed for genome editing. CRISPR-Cas9 can be programmed with a synthetic single guide RNA (sgRNA), enabling it to bind and cut a genomic target at any desired location. This way genes can be removed or replaced. Potential applications include curing genetic diseases, crop improvement and the development of biological platforms that can produce various compounds [3]. The simplicity of the CRISPR-Cas9 technique has made genome-editing so simple that it can, literally, be done at home by untrained personnel [4]. With great power, comes great responsibility. In fact, the simplicity and costs of this particular genetic modification technique raised questions about the consequences for biosecurity and, if it could be used to create novel bioweapons [5]. An exploitable novel type of bioweapons based on CRISPR-Cas9 is, for example, the use of gene drives which would allow the genome editing of an entire population. A gene drive [6] is a genetic element that increases its own chance of being inherited by ensuring homozygosity. To initiate a gene drive, the CRISPR-Cas9 gene needs to be implemented in the host genome [7]. Cas9 is utilized to edit, remove or add genes and facilitates the introduction of an additional copy of the Cas9 gene on the other chromosome. The organism will be, thus, homozygotic for the mutation and the Cas9 gene, and will pass it on to the entire offspring. This effect can be used to spread a genetic modification quickly through a population (Figure 1). Figure 1. Schematic representation of gene drive spreading through generations. Green dots show gene drive carrying individuals and grey dots represent wild type organisms. Heterozygous individuals carrying the gene drive will become homozygous allowing the mutation to rapidly spread through a population. Numerous applications have been envisioned which use a gene drive to solve global agricultural, medical and environmental problems. For example, agricultural and ecological pests can be eradicated by modifying the evolutionary fitness or propagation traits of certain species [8]. Furthermore, it would be possible to use gene drive technology to eliminate hosts carrying vector-borne diseases such as Malaria, Zika or Dengue [9]. The potential of gene drives to alter mosquitoes so that they will no longer spread human diseases or to restore ecosystems has been widely discussed [10]. But more sinister applications have been identified as well: altering pests so they will start to spread human diseases, making agricultural pests unsusceptible to conventional pesticides or the disturbance of ecosystems by targeting essential organisms [11]. In fact, after the initial proof of principle of gene drive technology in 2015, 26 scientists published a letter in the journal Science calling for safety measures for experimenting with gene drive, even in a laboratory setting [12]. Even an accidental outbreak can have far- reaching consequences for the ecosystem. The possibility of using gene drives on purpose as a bioweapon is even more worrisome. For this reason, scientists have already started to design procedures to undo gene drives [13]. However, the first step, before initiating a security protocol or undoing a gene drive, lies in the detection of gene drives in the host organisms. Time is essential when dealing with gene drives, as mutations must be detected before the modified genetic element has time to spread through the population. Conventional methods of detection are time-consuming, because they require transport of organisms or specimens to a lab. Furthermore, analysis techniques like sequencing are time- consuming and expensive. MinION, a portable sequencing device, decreased the time of sequencing and the price with respect to more lab-oriented instruments. However, the DNA must be purified before sequencing analysis, making this methodology rather cumbersome [14] and not applicable in-field. Hence, a quick and easily operated screening method is required that can detect the presence of a gene drive immediately in the field. A DNA amplification method can be used as detection method, as it will amplify the specific target sequence only if the target DNA is present. This leaves the question what DNA should be targeted, because CRISPR-Cas9 modifications by themselves do not leave traces in the modified genome. Fortunately, gene drives have an important difference compared to a normal (induced) CRISPR Cas9 mutation: a gene drive can only function if the endonuclease itself is embedded within the host genome. This means that the presence of a foreign endonuclease is an excellent marker to identify whether or not a gene drive has been introduced in an organism. We therefore envision a detection method based on the amplification of the Cas9 endonuclease sequence inserted in the genome. One classic example of a DNA amplification method for detection of specific DNA sequence is the Polymerase Chain Reaction (PCR) [15]. In our approach, we employ another amplification technique known as Loop-Mediated Isothermal Amplification (LAMP) [16] to amplify the Cas9 endonuclease sequence. LAMP is more specific, cheaper and quicker than the conventional polymerase chain reaction (PCR) [16]. In addition, the LAMP reaction is isothermal, making it easier to use for an in-field screening method. To perform the LAMP reaction in the field, a cheap battery-operated Arduino system containing temperature-controlled reaction cells can be used. In addition, a quick visualization by naked eye is needed to verify the outcome of the amplification of the target sequence. Thus, if the organism contains a CRISPR-Cas9 gene drive element, the target sequences will be amplified and detected. In this research we show our approach for the in-field detection of CRISPR-Cas9 gene drive modified organisms. It consists of three components: 1) a suitable LAMP primer set that detects the proper target sequence; 2) a battery-operated and cheap device that can set and maintain the temperature at which the LAMP reaction takes place and 3) a visualization method that allows for a quick read-out of the outcome without the need of additional laboratory equipment. Material and methods: LAMP primers and LAMP reaction We designed multiple primer sets for the Cas9 sequence from Streptococcus pyogenes (also called SpyCas9) using Primer Explorer version 5. Each primer set consisted of six primers. All designs were checked for primer dimerization, unwanted possible side reaction for the LAMP, using Thermofischer Tool. The specificity of the primers was checked using NCBI primer blast software. A g-block of 343 base pairs from the S. pyogenes Cas9 sequence was ordered from IDT. Based on dimerization and specificity results, one set of six primers was ordered and tested for nonspecific amplification by measuring melting curve temperatures using real-time LAMP Genie II instrument with a LAMP reaction mixture from OptiGene. The sequences and reaction concentration of the primers can be found in table 1.
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