Engineering Success
Our team aimed to design and optimize a CRISPR-Cas9 system to target the mraY and dnaG genes of Staphylococcus aureus, two essential genes involved in cell wall synthesis and DNA replication, respectively. The engineering process was iterative and guided by the Design–Build–Test–Learn (DBTL) framework, allowing us to refine each stage from DNA extraction to in vitro gene cleavage.
CYCLE 1: Genomic DNA Extraction and PCR Amplification
Design
We began by planning to amplify the mraY and dnaG genes from S. aureus genomic DNA for downstream CRISPR-Cas9 assays. Primers were designed to flank the target regions and optimized for GC content and melting temperature.
Build
Genomic DNA was extracted from S. aureus cultures and used as a template for PCR amplification of mraY (~2.5 kb) and dnaG (~4.9 kb).
Test
Initial PCR reactions failed due to insufficient or degraded genomic DNA. After improving extraction protocols, faint or smeared bands appeared across temperature gradients (59.6°C–60.9°C).
- mraY: Weak non-specific amplification
- dnaG: No amplification, likely due to secondary structure or long fragment size
Learn
We discovered that:
- The DNA template quality was critical for successful amplification.
- Large fragments such as dnaG may require preheating steps to denature secondary structures.
- Annealing temperature optimization was not enough; DNA concentration and primer specificity also needed refinement.
CYCLE 2: Optimization of PCR and In Vitro CRISPR-Cas9 Cleavage
Design
Based on previous results, we redesigned the PCR workflow to include DNA precipitation and purification steps using sodium acetate and isopropanol. In parallel, we planned in vitro Cas9–gRNA cleavage assays to validate targeting efficiency.
Build
Purified genomic DNA was used for re-amplification:
- mraY successfully yielded a ~2.5 kb band after optimization.
- dnaG amplification was achieved after re-amplification, though the band appeared larger (~4.9 kb), suggesting non-specific amplification.
For in vitro cleavage, Cas9 and gRNA concentrations were optimized across multiple ratios:
- Initial ratio: 50 nM gRNA + 12.5 nM Cas9
- Modified ratio: 10 nM Cas9 + 20 nM gRNA (1:2 and 1:5 tested)
Test
Cleavage efficiency varied with different Cas9:gRNA ratios.Results indicated that:
- Lower DNA concentrations and sufficient Cas9 levels were essential for complete cleavage.
- Excess gRNA could lead to dimerization, reducing binding efficiency with Cas9.
- High DNA input required more Cas9 to achieve full cleavage.
Learn
We concluded that:
- The ratio between Cas9, gRNA, and DNA must be carefully balanced.
- Optimal cleavage occurred at low DNA, high Cas9, and moderate gRNA levels.
- Our modified protocol (10 nM Cas9 + 20 nM gRNA, ratio 1:2) provided the most consistent cleavage pattern.