CRISPRi

Overview

Problem

Antimicrobials have been the primary treatment for various infections caused by bacteria, fungi, and certain parasites. However, the overuse of antimicrobials has created an evolutionary selective pressure that favors the development of resistance among species of pathogenic organisms (Khalid Ahmed et al., 2024). This has led to the emergence of antimicrobial resistance (AMR), where organisms are less or entirely unresponsive to original medications (WHO, 2023). With AMR, infections are increasingly difficult to treat, with efforts to rapidly develop new antibiotics and optimize existing ones stagnating (Salam et al., 2023). Therefore, Lambert iGEM created a rapidly adaptive therapeutic for AMR using CRISPR-interference (CRISPRi), as an alternative to traditional antimicrobials.

Methodology

Lambert iGEM pursued CRISPR-interference (CRISPRi) to downregulate critical genes in pathogens. CRISPRi utilizes a deactivated Cas9 (dCas9) endonuclease and specific oligonucleotide single-guide RNA (sgRNA) to create a dCas9-sgRNA complex (see Fig. 1)(Larson et al., 2013). This complex inhibits the transcription of the critical gene by binding to it and preventing RNA polymerase from continuing transcription (Marshall et al., 2018). By precisely engineering this mechanism to target genes associated with an organism’s pathogenicity and survival, we have developed a promising new antimicrobial.

Figure 1. Diagram of the CRISPRi Mechanism

To express dCas9 and sgRNA constructs in vitro, we decided on utilizing myTXTL (cell free transcription-translation) lysates from Daciel Arbor Biosciences, enabling the rapid expression of both linear and plasmid DNA. This approach allowed us to eliminate the need for bacterial transformation and in vivo testing, increasing the safety and efficiency of our experiments.

Alternative to Traditional Antimicrobials

AMR develops through mutations that render certain antibiotics ineffective, typically requiring the costly and time-consuming development of new antibiotic classes—a sector that has stagnated due to resource constraints. CRISPRi, however, offers a more adaptable solution. By allowing researchers to rapidly design new guide RNAs targeting alternative genes, CRISPRi circumvents the challenge of mutated targets without the need for entirely new antibiotics. This approach not only saves considerable time and money in drug development but also enables the medical industry to swiftly respond to emerging resistance, providing a powerful tool in the ongoing battle against AMR. Additionally, it allows for researchers to develop antimicrobials for any disease, due to the versatility of CRSIPRi and its ability to effectively silence any target gene.

Justification

Our team chose CRISPRi for its various advantages over other methods of gene repression and other potential antimicrobial therapies. For one, CRISPRi provides enhanced safety compared to traditional CRISPR-Cas9 systems, as it does not make permanent genomic alterations, helping to avoid potential biosafety hazards (Li et al., 2023). Additionally, CRISPRi offers greater specificity and higher on-target effects compared to other genomic editing technologies like RNA interference (RNAi) (Boettcher & McManus, 2015).

CRISPR vs CRISPRi

The traditional CRISPR-Cas9 system uses a Cas9 protein to cut double-stranded DNA at specific locations in the genome. After cleavage, the repair of double strand breaks relies on processes like nonhomologous end joining (NHEJ) and homology-directed repair (HDR) to restore the DNA by connecting the ends of the cleaved sequences. However, these systems are highly error-prone, often leading to unintended mutations and genomic instability (Yang et al., 2020). In contrast, CRISPRi does not rely on these repair systems, instead using dCas9 and sgRNAs to bind to target DNA sequences without inducing double-strand breaks. This method avoids unintended genetic alterations, as the dCas9-sgRNA complex naturally dissociates from target genes over time, ensuring no permanent genetic changes occur. By avoiding the need for DNA cleavage and repair mechanisms, CRISPRi offers a more controlled and reliable option for gene regulation(Qi et al., 2013). Its non-permanent modification addresses critical biosafety concerns associated with lasting genomic changes, which is particularly crucial in high school laboratory settings (Qi et al., 2013). The ability for CRISPRi to temporarily modify the genome minimizes the risk of unintended consequences, such as the accidental release of modified organisms into the environment, making it a safer option overall (Zhang et al., 2021). Furthermore, because antimicrobial resistance primarily stems from random mutations within pathogen genomes, error-prone gene editing mechanisms like traditional CRISPR may increase the likelihood of mutations and the development of resistance. (Marraffini & The Rockefeller University, 2021). CRISPRi does not edit the genome nor are its effects permanent, making it a more suitable method of targeting AMR (see Fig. 2).

Figure 2. Comparison between traditional CRISPR and CRIPSRi

RNAi vs CRISPRi

RNA interference (RNAi) uses small interfering RNAs (siRNAs) to degrade target mRNA, effectively silencing gene expression post-transcriptionally (Boettcher & McManus, 2015). Although RNAi is effective at reducing gene expression, it can result in off-target effects and variable gene silencing efficiency. These off-target effects occur when siRNAs partially complement other mRNAs besides the target. This imperfect binding causes siRNA perturbation, leading to unintended gene silencing (Dua et al., 2011). In contrast, research has shown that CRISPRi can silence target genes up to 99.9% repression. Due to its simplicity in design, CRISPRi is likely to yield more specific results compared to RNAi (See Fig. 3)(Larson et al., 2013).

Figure 3. Diagram of CRISPRi and RNAi

References