Senior Director, IBC Services
WCG Clinical
Advanced engineered cell therapies require gene editing tools that are both precise and efficient. In recent years, CRISPR-Cas9 has emerged as the gold standard for editing genes with greater precision than older techniques. Unlike viral vectors that randomly integrate DNA into the genome, Cas9 and similar endonucleases cut DNA at specific sites, allowing for controlled modifications. However, CRISPR-Cas9’s full potential in clinical applications remains limited by a fundamental challenge: getting enough Cas9 protein into the cell nucleus, where it can reliably modify cellular DNA.
Most current approaches rely on adding nuclear localization signal (NLS) motifs to the ends of Cas9 to facilitate nuclear entry. However, this method is inefficient, and much of the Cas9 that is provided to cells never reaches the nucleus. Addressing this inefficiency is critical for successful therapeutic applications, where any enhancement could mean more effective treatments.
A recent publication by researchers at the University of California Berkeley’s Innovative Genomics Institute unveils a clever solution to boost Cas9’s nuclear entry—and thus its gene editing performance—by increasing the number of NLS motifs within the Cas9 protein.1 Here, I highlight how this novel approach improves gene editing in human T cells and discuss its implications for the development of future cell therapies.
A new approach to nuclear delivery
Traditional Cas9 designs typically include one to three NLS motifs attached to the protein’s C- and N-termini. The Berkeley team hypothesized that adding more NLS motifs to different areas of Cas9 could improve nuclear import, but simply extending the terminal tails with additional NLS motifs proved problematic. In line with prior experimental data, a Cas9 with six terminal NLS motifs showed poor expression yields, making it impractical for large-scale production.
Their innovative solution was to insert additional NLS motifs into internal loops of the Cas9 protein, where they would be more evenly distributed across the protein, rather than at C- or N-termini, which are located closely together in the protein’s 3D structure.2 By analyzing the protein’s structure, they identified several surface-exposed loops where NLS insertions would be tolerated, allowing for the addition of NLS without compromising the protein’s stability or activity.
Each hairpin internal NLS (hiNLS) module consists of two NLS motifs arranged in tandem, separated by flexible linkers. This tandem design ensures that if one motif temporarily detaches from the importin proteins that mediate nuclear entry, the other can still hold on, increasing the likelihood that Cas9 remains bound during transit into the nucleus. The team generated 15 different Cas9 variants with one to four hiNLS modules inserted, yielding variants with up to nine individual NLS motifs.
Testing in primary human T cells
To evaluate their hiNLS-Cas9 variants, the researchers tested their ability to edit primary human T cells, which are a cornerstone of many cell therapies, including chimeric antigen receptor (CAR) T cells. The team introduced gRNA-Cas9 ribonucleoprotein complexes (RNPs) targeting two clinically relevant genes: b2M (beta-2 microglobulin) and TRAC (T-cell receptor alpha chain). Disrupting b2M helps create immune-evasive cell therapies by eliminating expression of major histocompatibility complex I (MHC-I) molecules on T cells, while loss of TRAC can prevent graft-versus-host reactions and make space for synthetic receptors in CAR T cells.3
The researchers delivered RNPs to cells using two methods: standard electroporation and a peptide-mediated delivery method (peptide-enabled ribonucleoprotein delivery for CRISPR engineering [PERC]), which relies on peptide-RNP complexes to facilitate cell entry.4 Compared to electroporation, PERC is gentler and simpler, with less impact on cell viability and requiring no specialized equipment. The study’s authors therefore used PERC as a surrogate for in vivo editing technologies—such as lipid nanoparticles or virus-like particles—where preserving cell viability is critical and the editing window is limited due to transient RNP availability.
When multiple hiNLS modules were combined in one Cas9, editing rates improved significantly. For example, a Cas9 with two NLS module inserts (s-M1M4) delivered via electroporation knocked out the b2M gene in over 80% of T cells, compared to about 66% with traditional Cas9. Using the PERC method, several multi-hiNLS constructs achieved 40–50% knockout efficiency, whereas the control managed around 38%. Throughout testing, T-cell viability after editing remained unaffected by the extra NLS, which is reassuring for therapeutic applications. Interestingly, a direct association between the number of NLS inserts and editing efficiency was not observed. However, variants rich in c-Myc-derived NLS outperformed those using SV40 NLS signals, suggesting that NLS sequence quality matters at least as much as quantity.
Key advantages for the clinic
This study demonstrates several practical advantages of hiNLS-Cas9 for therapeutic development:
- Higher Editing Efficiency: By addressing the nuclear entry bottleneck, hiNLS-Cas9 can achieve higher editing percentages in T cells than conventional Cas9. This suggests other difficult-to-edit cell types could also benefit from an hiNLS boost, making both experiments and therapies more effective. PERC-based delivery of hiNLS-Cas9 variants resulted in editing efficiencies similar to those seen with electroporation. Since PERC is less cytotoxic than electroporation, this could mean even greater percentages of edited cells from the same amount of Cas9, also critical for scalability.
- Maintained Protein Yield: Unlike Cas9 with multiple terminal NLS motifs, the hiNLS variants remained easy to produce, with recombinant yields of 4-9 mg per liter, comparable to unmodified Cas9. This means labs and companies can manufacture hiNLS-Cas9 at scale without special techniques, which will be crucial for clinical translation.
- Rapid Action for Transient Delivery: The improved nuclear localization is especially valuable for transient delivery formats like RNPs or mRNA, where the editing window is brief. The results showed hiNLS-Cas9 can capitalize on that short window, potentially reducing the required dose or increasing editing achieved per dose.
For cell therapy manufacturing, where editing efficiency directly impacts production costs and timelines, these improvements could be transformative. More specifically, current CAR T manufacturing processes face challenges with consistency and yield, so higher editing rates could help address these issues.
Future directions and combinations
Combining the hiNLS-Cas9 method with parallel advances multiplies the possibilities. Scientists are exploring virus-like particles and lipid nanoparticles to deliver Cas9 RNPs directly into patients for in vivo editing.5 Such in vivo approaches often suffer from low delivery rates to the nucleus, but an NLS-boosted Cas9 might increase editing in the few that do arrive. Another complementary avenue is engineering Cas9 for higher specificity. The authors noted a slight uptick in off-target activity at one known problematic site when using hiNLS-Cas9, likely because the extra NLS motifs help Cas9 remain bound to DNA longer. Merging high-fidelity Cas9 mutations with hiNLS could yield an enzyme that is both accurate and highly efficient.6
Looking ahead, the hiNLS-Cas9 strategy could be applied to other genome editors, such as Cas12a or base editors, which face similar delivery constraints. This work also underscores an important design concept: rather than oversimplifying an enzyme with just terminal tags, we can rationally modify it from within to balance performance and practicality. As gene editing moves toward multiplexed approaches where multiple genes are targeted simultaneously, the efficiency of each individual edit becomes increasingly important. Higher per-target efficiency with hiNLS-Cas9 could make complex multiplex editing more feasible, opening doors to next-generation cell therapies with sophisticated engineered functions.
By focusing on the often-overlooked aspect of nuclear localization, researchers have created a more efficient tool that could help accelerate the development of gene-edited cell therapies while potentially reducing manufacturing costs—a win for both developers and patients awaiting these innovative treatments.
Christopher Doyle, PhD, is the senior director of IBC Services at WCG Clinical.
References
1. Noel EA, Sahu SU, Wyman SK, Krishnappa N, Jeans C, Wilson RC. Hairpin Internal Nuclear Localization Signals in CRISPR-Cas9 Enhance Editing in Primary Human Lymphocytes. CRISPR J. 2025;8(2):105-119. doi:10.1089/crispr.2024.0080
2. Jiang F, Taylor DW, Chen JS, et al. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage. Science. 2016;351(6275). doi:10.1126/science.aad8282
3. McCarty NS, Graham AE, Studená L, Ledesma-Amaro R. Multiplexed CRISPR technologies for gene editing and transcriptional regulation. Nat Commun. 2020;11(1). doi:10.1038/s41467-020-15053-x
4. Foss DV, Muldoon JJ, Nguyen DN, et al. Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes. Nat Biomed Eng. 2023;7(5). doi:10.1038/s41551-023-01032-2
5. Alsaiari SK, Eshaghi B, Du B, et al. CRISPR–Cas9 delivery strategies for the modulation of immune and non-immune cells. Nat Rev Mater. 2024;10(1):44-61. doi:10.1038/s41578-024-00725-7
6. Yin S, Zhang M, Liu Y, et al. Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies. Mol Ther. 2023;31(3):744-759. doi:10.1016/j.ymthe.2022.11.014
