CRISPRi
Therapeutic approach to combat AMR using CRISPR-interference
Design
CRISPR-interference
The CRISPR-interference (CRISPRi) mechanism combines the use of deactivated Cas9 (dCas9) and single-guide RNA (sgRNA) to repress specific genes. By providing sgRNAs that are complementary to target genes, the dCas9 protein and sgRNA form a dCas9-sgRNA complex that can accurately locate and downregulate these genetic sequences by preventing RNA polymerases from binding (see Fig. 1).
dCas9-sgRNA complex
For the dCas9-sgRNA complex to form, the sgRNA must first fold and create secondary structures. Once the scaffold region of the sgRNA forms a loop, the nucleotides interact with the dCas9 protein, causing a conformational change that allows dCas9 to bind to the DNA. A specific 15-20 nucleotide-long region known as CRISPR RNA (crRNA) is able to facilitate binding of the CRISPRi system to DNA, as crRNA is complementary to a part of target DNA (Synthego, 2019; Costa et al., 2017). Then, the trans-activating CRISPR RNA (tracrRNA) fuses with the crRNA to help guide the dCas9 to correctly bind with the target sgRNA site (see Fig. 2).
dCas9
Lambert iGEM utilized pCD017-dCas9 (BBa_K5096056), a type of dCas9 derived from Streptococcus pyogenes, which was obtained from Dr. Vincent Noireaux, a researcher at the University of Minnesota who specializes in cell free expression of the CRISPRi system. This specific dCas9 plasmid has been widely researched and used in CRISPRi systems, particularly in the TXTL cell-free system, due to its proven efficacy and the protocols outlined in the literature.
sgRNA
To develop a sgRNA for CRISPRi, we had to consider several constraints when selecting the target site where the sgRNA binds. The target site has to be within 100 nucleotides of a PAM site – a specific 3-nt long sequence – which the dCas9 will recognize, to ensure low off-target effects. Also, dCas9 recognizes NGG PAMs most effectively, and repression is stronger when targeting the nontemplate strand of target DNA (Marshall et al., 2018) Additionally, by using Benchling’s software, we can generate options for sgRNAs and predict the on-target and off-target effects exhibited by each sgRNA (Benchling, 2023). This enabled us to select sgRNAs for our testing that were predicted to have the highest on-target effects with the CRISPRi system, ensuring at least a 98% chance of not having off-target effects (see Fig. 4).
TXTL
Lambert iGEM selected commercial cell-free systems to test the CRISPRi mechanism due to the extensive existing research on CRISPRi expression in these systems (Gregorio et al., 2019; McGaw & Chong, 2021; ThermoFisher Scientific, 2024). Previously, researchers found that traditional assays for CRISPRi reactions required weeks to perform due to the need for protein purification or bacterial transformation, both of which produce low yields of protein expression. Given the increasing need for more efficient systems, researchers adopted cell-free transcription-translation (TXTL) systems. Among these, the cell-free TXTL system featured comprehensive documentation, allowing us to perform the CRISPRi reaction in vitro. Specifically, we used the myTXTL Sigma70 Master Mix Kit from Arbor Biosciences, due to its compatibility with the cell-free expression of the dCas9 enzyme essential for CRISPRi reactions; however, once the Sigma70 kits were discontinued, we transitioned to Arbor Bioscience’s myTXTL Pro Cell-Free Expression Kit (Marshall et al., 2018). The TXTL system is based on Escherichia coli lysates prepared from exponentially growing cells that preserve the natural transcriptional, translational, and metabolic machinery. This system allows DNA encoding genes of interest to be added to the lysates in different volumes, resulting in gene expression within hours. While plasmid DNA is commonly used, TXTL can also accommodate linear DNA, given that RecBCD - a protein that degrades dsDNA - is inhibited using GamS or Chi6 (Marshall et al., 2017). TXTL reactions can be conducted in volumes as small as a few microliters, allowing scalable use in 384 well plates. This approach allows for a more efficient utilization of linear DNA and small reactions, making it suitable for CRISPRi experiments.