Recent advancements in gene editing technology have revealed a promising method to enhance the efficiency of the CRISPR-Cas9 system in targeting human cells. Researchers from the University of California Berkeley’s Innovative Genomics Institute have introduced an innovative approach to improve the nuclear entry of Cas9 proteins, a crucial step for effective gene editing. The findings could significantly impact the development of advanced cell therapies.
CRISPR-Cas9 has established itself as a leading tool for precise gene editing, surpassing previous techniques due to its ability to cut DNA at specific sites. Traditional methods of delivering Cas9 into the cell nucleus often rely on adding nuclear localization signal (NLS) motifs. However, this strategy has shown limited success, as a significant portion of the Cas9 protein fails to enter the nucleus, hampering its therapeutic potential.
Innovative Approach to Nuclear Delivery
The Berkeley research team sought to resolve the inefficiencies associated with nuclear entry by increasing the number of NLS motifs in the Cas9 protein. Conventional Cas9 typically has one to three NLS motifs located at its ends. The researchers hypothesized that adding more NLS motifs throughout the protein could enhance its nuclear import.
Their findings revealed that merely extending the terminal tails with additional NLS motifs resulted in poor expression yields, making large-scale production impractical. Instead, they opted to insert extra NLS motifs into internal loops of the Cas9 protein. This internal placement allowed for a more effective distribution of NLS motifs without compromising the protein’s stability or functionality.
The team developed multiple Cas9 variants, incorporating hairpin internal NLS (hiNLS) modules that feature two tandem NLS motifs separated by flexible linkers. This design increases the likelihood that one motif will remain attached to importin proteins during nuclear transport, thus enhancing the overall effectiveness of Cas9 delivery.
Testing the hiNLS-Cas9 Variants
To assess the performance of their hiNLS-Cas9 variants, the researchers conducted experiments on primary human T cells, which are central to various cell therapies, particularly chimeric antigen receptor (CAR) T cell therapy. They targeted two clinically relevant genes: b2M (beta-2 microglobulin) and TRAC (T-cell receptor alpha chain). Disruption of the b2M gene is crucial for creating immune-evasive cell therapies, while loss of TRAC can help prevent graft-versus-host reactions.
The team employed two delivery methods to introduce gRNA-Cas9 ribonucleoprotein (RNP) complexes into the T cells: standard electroporation and a gentler peptide-mediated delivery method known as PERC. Compared to electroporation, which can negatively affect cell viability, PERC provides a more straightforward and less invasive approach.
In their tests, Cas9 variants with multiple hiNLS modules demonstrated significantly improved editing rates. For instance, a specific variant with two NLS module inserts achieved over 80% knockout efficiency of the b2M gene in T cells through electroporation, surpassing the traditional Cas9’s effectiveness of around 66%. Using the PERC method, some multi-hiNLS constructs reached 40-50% knockout efficiency, compared to approximately 38% with the control.
Importantly, the introduction of additional NLS motifs did not compromise T-cell viability, an essential factor for therapeutic applications. While the study did not establish a direct correlation between the number of NLS inserts and editing efficiency, variants with c-Myc-derived NLS signals outperformed those using SV40 NLS sequences, indicating that the quality of NLS sequences may be as crucial as their quantity.
The research highlights several practical advantages of the hiNLS-Cas9 system for clinical applications. By improving nuclear entry, this method has the potential to increase the editing efficiency of T cells, which could also extend to other challenging-to-edit cell types. The successful use of PERC delivery for hiNLS-Cas9 variants suggests that therapies could achieve higher editing rates without compromising cell viability.
Additionally, the hiNLS variants maintained protein yields comparable to unmodified Cas9, making them easier to produce in large quantities. This aspect is vital for laboratories and companies aiming to scale up production for clinical uses.
As gene editing continues to evolve, the integration of hiNLS-Cas9 with other emerging technologies presents exciting possibilities. Researchers are investigating the use of virus-like particles and lipid nanoparticles for direct in vivo delivery of Cas9 RNPs. By enhancing NLS-mediated nuclear delivery, the hiNLS approach may increase the effectiveness of these in vivo strategies.
Looking ahead, the potential to apply the hiNLS-Cas9 method to other genome editing systems, such as Cas12a or base editors, could further address similar delivery challenges. This advancement emphasizes the need for innovative design concepts that enhance the efficiency of gene editing.
By focusing on optimizing nuclear localization, the Berkeley team’s research offers a promising pathway to accelerate the development of gene-edited cell therapies while potentially reducing manufacturing costs. This advancement could represent a significant step forward for both developers and patients awaiting access to cutting-edge treatments.
