Droplet based microfluidics
CRISPR, short for “Clustered Regularly Interspaced Short Palindromic Repeats,” is a naturally occurring mechanism found in bacterial immune systems, aiding in defense against viral invaders. Scientists have leveraged this system as a genome editing tool to modify genes within living cells. CRISPR are repetitive DNA sequences found in the genomes of bacteria and archaea, interspersed with segments of DNA known as spacers, which are derived from previous viral or plasmid infections 1.
Cas9 (CRISPR-associated protein 9) is an enzyme that acts as a molecular scissors, capable of cutting DNA at specific locations guided by a small piece of RNA called guide RNA (gRNA). The gRNA is designed to complement a target DNA sequence, guiding Cas9 to precisely cleave the DNA at that location. When Cas9 binds to the target DNA sequence guided by the gRNA, it induces a double-strand break (DSB) in the DNA. This break can then be repaired by the cell’s own DNA repair machinery through either non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ often results in small insertions or deletions (indels) at the site of the break, leading to gene disruptions or mutations (Figure 1). HDR, on the other hand, can be used to introduce specific changes or edits to the DNA sequence by providing a repair template along with the CRISPR-Cas9 system 2 .
Cas9 has the capability to initiate precise double-strand breaks (DSBs) in target DNA via a dual-RNA-guided mechanism. This process involves the fusion of crRNA with its complementary trans-activating crRNA (tracrRNA), directing Cas9 to the target sequence (Figure 2). A prerequisite for Cas9 action is the presence of a proto-spacer-adjacent motif (PAM) sequence (typically NGG), which initiates the recognition process. Upon PAM recognition, the spacer region of the guide RNA pairs with the target DNA strand, forming an “R-loop” structure, ultimately leading to DNA cleavage three base pairs upstream of the PAM site. Cas9 harbors HNH and RuvC nuclease domains, responsible for cleaving the complementary and noncomplementary DNA strands, respectively. Modifications such as inactivating one or both of these domains yield variants like Cas9 nickase (nCas) or dead Cas9 (dCas9), which retain DNA binding capability but lack nuclease activity 4.
One of the primary challenges in CRISPR applications is off-target effects, where Cas9 may cleave unintended sites due to mismatches between the guide RNA and off-target DNA. Various strategies have been devised to mitigate this issue, including modifying guide RNAs, engineering the Cas9 protein, and screening for novel enzymes. For instance, truncated guide RNAs (Tru-gRNAs), hairpin-structured sgRNAs (hp-sgRNAs), and CRISPR hybrid RNA-DNA (chRDNA) guides have been developed to enhance Cas9 specificity while maintaining on-target activity. Additionally, techniques like utilizing paired Cas9 nickases or fusing dCas9 with the FokI nuclease domain have shown promise in improving specificity. Furthermore, the exploration of high-fidelity Cas9 variants through rational design or directed evolution offers potential solutions for enhancing specificity in diverse applications 6.
Despite its robust activity, Cas9’s reliance on specific PAM sequences poses limitations on its flexibility and utility. Efforts are underway to develop PAM-free nucleases through natural ortholog exploration and protein engineering, aiming to expand the scope of CRISPR applications. These advancements hold promise for addressing current challenges and broadening the applicability of CRISPR-based technologies in various research and practical contexts. The discovery of the RNA-guided DNA nuclease CRISPR-Cas9 has facilitated precise genome editing across various organisms. However, the permanent and heritable alterations to the genome raise significant concerns regarding potential risks. These risks include accidental exposure, deliberate misuse, and the persistence of Cas9 therapeutics, as well as off-target effects on the genome. Therefore, the development of detection assays is crucial 7.
The CRISPR-Cas system functions through three main stages: adaptation, expression, and interference (Figure 3), with the ultimate goal of targeting and degrading invading viruses or plasmids. Here’s an explanation of each stage 8:
In this stage, the CRISPR-Cas system acquires small segments of foreign DNA from invading viruses or plasmids and incorporates them into the bacterial genome as “spacer” sequences. This process is facilitated by specific enzymes within the system. These spacer sequences serve as a memory of past infections and allow the system to recognize and respond to similar invaders in the future.
Once the spacer sequences are integrated into the bacterial genome, they are transcribed into short RNA molecules known as CRISPR RNAs (crRNAs). Additionally, the CRISPR locus contains repeat sequences that are transcribed into precursor CRISPR RNAs (pre-crRNAs). These pre-crRNAs are then processed into mature crRNAs, which consist of the spacer sequence along with a portion of the repeat sequence.
In the interference stage, the CRISPR-Cas system utilizes the crRNAs to guide Cas proteins (such as Cas9) to complementary sequences in foreign DNA or RNA molecules. When the crRNA binds to a matching sequence, the Cas protein undergoes a conformational change, leading to the activation of its nuclease activity. This results in the cleavage or degradation of the invading genetic material, ultimately preventing the virus or plasmid from replicating and infecting the bacterial cell.
Overall, the adaptation stage allows the CRISPR-Cas system to acquire a memory of past infections, the expression stage involves the transcription and processing of CRISPR RNAs, and the interference stage utilizes these RNAs to guide Cas proteins to target and degrade foreign genetic material, providing a defense mechanism against viral and plasmid invasion.
CRISPR-Cas9 has found widespread use in various fields, from basic research to therapeutic applications, due to its simplicity, versatility, and high efficiency. However, the manual-intensive nature of traditional CRISPR workflows presents challenges in terms of scalability, consistency, and throughput. CRISPR-Cas9 allows researchers to edit genes with unprecedented precision and ease, enabling a wide range of applications such as studying gene function, creating disease models, developing new therapies, and potentially treating genetic disorders in humans. Additionally, CRISPR-Cas9 has implications beyond research, with potential applications in agriculture, biotechnology, and medicine, promising significant advancements in various fields3.
Currently, microfluidics stands as a prominent and clearly delineated technological domain. However, two decades ago, the historical landscape of microfluidics lacked such clearly established boundaries. Microfluidics technology represents a significant advancement in the field of biotechnology, offering precise control over the manipulation of small volumes of fluids on a microscale. However, despite its tremendous potential, there are several challenges and considerations associated with the widespread adoption and implementation of microfluidics technology, leading to what can be termed an “overflow” of challenges and opportunities. One of the primary challenges is the complexity of microfluidic device fabrication and operation. Designing and fabricating microfluidic devices often require specialized equipment and expertise, which can be a barrier to entry for researchers and industries unfamiliar with the technology. Additionally, the integration of multiple components and the optimization of fluidic parameters can be technically demanding, requiring careful calibration and validation 10.
Microfluidics technology offers a promising solution to these challenges by enabling precise control and manipulation of small volumes of fluids on a microscale. Microfluidic devices can integrate various steps of the CRISPR workflow, such as guide RNA delivery, cell transfection, and clone selection, into a single automated platform. By miniaturizing and parallelizing these processes, microfluidics can significantly increase the efficiency and throughput of CRISPR experiments while reducing reagent consumption and labor costs. The integration of CRISPR-Cas9 with microfluidics has led to the development of innovative techniques for genetic editing and screening. For example, microfluidic devices can enable high-throughput screening of CRISPR libraries to identify gene targets involved in specific biological processes or diseases (Figure 4). Additionally, microfluidic systems can provide a controlled environment for cell culture and manipulation, leading to more reproducible and reliable results 11.
These devices can also facilitate the generation and selection of edited cell lines with desired genomic modifications, such as single-cell cloning and expansion. Furthermore, microfluidic-based systems offer the potential for real-time monitoring of CRISPR editing outcomes, allowing researchers to optimize experimental conditions and improve editing efficiency 11.
Phaneuf and colleagues 13 presented a centrifugal microfluidic platform designed for assessing both Cas9 protein levels and nuclease activity (Figure 5) 7. Given that Cas9 proteins from different bacterial species are utilized in biotechnology applications, this platform is capable of detecting Cas9 from commonly used species such as Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitidis, and Streptococcus thermophilus, utilizing readily available antibodies. Additionally, researchers demonstrate the use of the phage-derived anti-CRISPR protein AcrIIC1 as a capture reagent, which extends the detection capability to a broader range of species by binding to Cas9 from multiple sources. Since genome modification typically involves Cas9 nuclease activity, researchers integrated a fluorescence-based sedimentation nuclease assay into this platform. This assay enables the sensitive and simultaneous measurement of both Cas9 protein levels and activity within a single biological sample. By combining protein detection and nuclease activity assessment, this platform offers a comprehensive approach to monitoring CRISPR-Cas9 activity, thereby addressing important safety concerns associated with genome editing technologies.
Iwai and colleagues 14 introduced a novel microfluidic setup designed for CRISPR-based gene editing and high-throughput screening directly on a chip. This microfluidic device comprises a grid of 100 elements, each equipped with electrodes for two key operations: electrowetting to merge droplets and mix reagents, and electroporation for genetic transformation (Figure 6). With the ability to conduct up to 100 genetic modification reactions concurrently, this system offers a scalable approach for generating numerous engineered strains essential for systematically optimizing genetic pathways and predictable bioengineering. To illustrate its functionality, they conducted CRISPR-based modifications on two specific targets: first, disrupting the galactokinase enzyme (galK) function in Escherichia coli, and second, enhancing indigoidine production in E. coli by targeted engineering of the glutamine synthetase gene (glnA) and the blue-pigment synthetase gene (bpsA).
Overall, the combination of CRISPR-Cas9 and microfluidics holds tremendous promise for advancing genetic research and biotechnology applications. By automating and miniaturizing key steps of the CRISPR workflow, microfluidic devices can streamline experimentation, increase throughput, and improve reproducibility. As these technologies continue to evolve and become more accessible, they are likely to play an increasingly important role in driving innovations in genome editing, drug discovery, and personalized medicine.
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