Report on Major Instruments for Genome Engineering Using CRISPR

CRISPR-based genome engineering relies on a suite of advanced instruments to design, deliver, validate, and analyze gene edits. These instruments are critical for ensuring precision, efficiency, and reproducibility in CRISPR experiments. This CRISPR Forum Report focuses on the key instruments used in CRISPR research, highlighting their roles and applications.

Instruments for CRISPR Component Design and Synthesis

Oligonucleotide Synthesizers: These instruments synthesize guide RNAs and single-stranded DNA templates for homology-directed repair. Examples include MerMade Synthesizers by BioAutomation and AKTA Oligopilot by Cytiva. They are used for high-throughput synthesis of gRNAs and custom oligonucleotides.

DNA Sequencers: DNA sequencers verify the sequence of synthesized gRNAs, repair templates, and target DNA regions. Examples include Illumina MiSeq/NovaSeq and Oxford Nanopore MinION. They are essential for quality control and sequencing of target loci to confirm edits.

Instruments for CRISPR Delivery

Electroporation Systems: Electroporation systems deliver CRISPR components into cells by creating transient pores in the cell membrane. Examples include the Neon Transfection System by Thermo Fisher Scientific and the Gene Pulser Xcell by Bio-Rad. They are particularly useful for hard-to-transfect cells and high-throughput delivery.

Microinjection Systems: Microinjection systems directly inject CRISPR components into single cells or embryos. Examples include the Eppendorf FemtoJet and the Narishige IM-300 Microinjector. They are used for precise delivery into zygotes and single-cell studies.

Viral Vector Production Systems: These systems produce viral vectors for stable delivery of CRISPR components. Examples include the Lenti-X Packaging System and the AAVpro Helper Free System by Takara Bio. They are used for in vivo and ex vivo delivery and stable integration of CRISPR components.

Instruments for CRISPR Validation and Analysis

Next-Generation Sequencing Platforms: NGS platforms analyze the outcomes of CRISPR edits, including on-target and off-target effects. Examples include the Illumina NovaSeq 6000 and the PacBio Sequel IIe. They are used for whole-genome sequencing and amplicon sequencing to validate specific edits.

Digital PCR Systems: Digital PCR systems quantify the efficiency of CRISPR edits and detect low-frequency mutations. Examples include the Bio-Rad QX200 Droplet Digital PCR and the Thermo Fisher QuantStudio 3D Digital PCR. They provide precise quantification of edit rates and detection of rare off-target events.

High-Content Imaging Systems: High-content imaging systems visualize and quantify the effects of CRISPR edits at the cellular level. Examples include the PerkinElmer Opera Phenix and the Molecular Devices ImageXpress Micro Confocal. They are used for live-cell imaging and high-throughput phenotypic screening.

Flow Cytometers: Flow cytometers analyze and sort cells based on CRISPR-induced changes. Examples include the BD FACSAria Fusion and the Beckman Coulter CytoFLEX. They are used for enrichment of edited cells and multiparametric analysis of CRISPR outcomes.

Instruments for Functional Genomics and Screening

Automated Liquid Handling Systems These systems enable high-throughput preparation and delivery of CRISPR components. Examples include the Tecan Freedom EVO and the Beckman Coulter Biomek i7. They are used for CRISPR library screening and large-scale transfection experiments.

Microplate Readers: Microplate readers measure CRISPR-induced changes in cellular assays. Examples include the BioTek Synergy H1 and the Tecan Spark. They are used for quantification of reporter gene expression and high-throughput screening of CRISPR libraries.

Instruments for Structural and Mechanistic Studies

Cryo-Electron Microscopy: Cryo-EM studies the structure and mechanism of CRISPR-Cas complexes at near-atomic resolution. Examples include the Thermo Fisher Titan Krios and the JEOL CryoARM 300. They are used for structural analysis of Cas proteins and gRNA-DNA interactions.

Surface Plasmon Resonance Instruments: SPR instruments measure the binding kinetics of CRISPR components to target DNA. Examples include the Biacore 8K by Cytiva and the Sierra Sensors SPR-2. They are used for characterization of gRNA-DNA binding affinity and optimization of CRISPR components for specificity.

The study and application of CRISPR-based genome engineering rely on a diverse array of instruments, each playing a critical role in the design, delivery, validation, and analysis of gene edits. From oligonucleotide synthesizers and electroporation systems to NGS platforms and cryo-EM, these instruments enable researchers to achieve precise and efficient genome editing. As CRISPR technology continues to evolve, advancements in instrumentation will further enhance its capabilities and expand its applications in research and therapy.