Improving CRISPR/Cas9 Genome Editing with Single-Stranded DNA Payloads

CRISPR/Cas9 enables targeted genomic insertions, clearing the way for precise therapies for cancer, rare diseases, and more. One key advantage of targeted integration is conserving regulatory mechanisms controlling the expression of genes of interest. For example, investigators have found that targeting CAR constructs for insertion within the TRAC locus (T cell Receptor Alpha chain Constant region) leads to enhanced CAR-T cell potency and persistence (Eyquem et al. 2017).

T cell immunotherapies, such as CAR and TCR T cells, offer unparalleled opportunities for cancer patients. To make T cell therapies safer and more efficacious, scientists are developing innovative non-viral DNA payloads. Such is the case with the tools and approaches being developed by the Alexander Marson laboratory at UCSF, a leader in CRISPR/Cas9 based T cell genome editing.

Previously Marson’s lab had successfully established the use of CRISPR/Cas9 and guide RNA ribonucleoprotein complexes (RNPs), directly electroporated into T cells ex vivo, as an efficient gene editing strategy to develop TCR T cells. By following this strategy, Marson’s team has found that the format of the DNA payload critically determines insertion outcomes. For example, double-stranded DNA (dsDNA) can be efficiently knocked-in through co-electroporation with RNPs, especially when containing a Cas9 targeting sequence (CTS) (Nguyen et al. 2020). Nevertheless, the team found that dsDNA payloads also lead to more significant cellular toxicity than single-stranded DNA (ssDNA). Therefore, to harness the CTS-nuclear shuttling benefits while reducing toxicity associated with dsDNA payloads, Marson’s team has engineered a new approach consisting of a hybrid homology-directed repair DNA payload, which they refer to as a ssCTS template (Shy et al. 2021).

Learn more about the new Hybrid ssDNA Homology-Directed Repair Template (HDRT) designs incorporating Cas9 target sites (CTS) and their performance across a broad range of loci and cell types.

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Newly designed ssCTS templates containing two CTS domains, annealed through the use of complementary oligos, improved knock-in efficiency for ssDNA payloads. Marson’s team found that they could achieve close to 40% knock-in efficiency of longer sequences (~2.9kb), as exemplified by a B-cell maturation antigen (BCMA) specific CAR construct targeting the TRAC locus. Interestingly, upon further optimization, they discovered that only one CTS domain, annealed to the 5’ end of the long ssDNA payload, was sufficient to improve knock-in efficiency. The knock-in efficiency of optimized ssCTS payloads could be further enhanced by using small molecules (e.g., Non-homologous End Joining or NHEJ inhibitors) known to enhance the homology-directed repair process. Through this combined approach, the team achieved over 90% knock-in efficiency for some ssCTS payloads in clinically relevant cell types (i.e., primary human T cells).

Fully non-viral CRISPR/Cas9-based strategies for T cell engineering present the opportunity to develop safer T cell therapeutics. Having optimized the design and application of ssCTS to support efficient insertions in primary T cells, Marson’s team is now developing a workflow for Good Manufacturing Practice (GMP) compliant T cell engineering. About the source of long ssDNA, Shy et al. shared: “We partnered with Genscript to develop a GMP-compatible process for ssCTS template generation. Encouragingly, Genscript templates encoding a BCMA-CAR knock-in were able to be manufactured at large scale and consistently outperformed our internally generated HDRTs, showing lower levels of toxicity and higher knock-in efficiencies for both ssCTS and dsCTS constructs."

CRISPR/Cas9 Based T Cell Therapies Advancing in Clinical Trials

Many T cell therapeutics, developed using CRISPR/Cas9 genome editing tools, are advancing through the early stages of clinical evaluation. Currently, there are over fifteen clinical studies evaluating CRISPR/Cas9-based T cell therapeutics for a broad range of malignancies. Among these, various clinical studies are evaluating CRISPR/Cas9 engineered T cells carrying targeted insertions at the TRAC locus (Sarkar and Khan 2021).

CRISPR/Cas9 Engineered T Cell Immunotherapeutics-Targeted TRAC Locus Integration

Sponsor Identifier T cell Therapeutic Stage Disease
Caribou Biosciences, Inc. NCT04637763 CB-010: CRISPR-Edited Allogeneic Anti-CD19 CAR-T Cell  Phase I Relapsed/refractory B cell non-Hodgkin lymphoma
CRISPR Therapeutics AG NCT04035434 CTX110: Allogeneic Anti-CD19 CAR-T cell immunotherapy Phase I B cell malignancies
CRISPR Therapeutics AG NCT04244656 CTX120: Anti-BCMA Allogeneic CRISPR-Cas9-Engineered T Cells  Phase I Multiple Myeloma
CRISPR Therapeutics AG NCT04438083 NCT04502446 CTX130: Allogeneic CD70-directed T-cell immunotherapy Phase I Renal Cell Carcinoma and T Cell Lymphoma
Fate Therapeutics NCT04629729 FT819: iPSC-derived T-cell–like cells expressing an anti-CD9-CAR transgene  Phase I Relapsed/refractory B-cell Lymphoma, Chronic Lymphocytic Leukemia, and Precursor B-cell Acute Lymphoblastic Leukemia
Great Ormond Street Hospital for Children NHS Foundation Trust NCT04557436  PBLTT52CAR19: Allogeneic engineered anti-CD19 T cells Phase I B Acute Lymphoblastic Leukemia


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