The use of CRISPR to construct CAR-T cells: A Strong Alliance!

Since Kymriah was approved for use as the global CAR-T “first drug” in 2017, CAR-T cell therapy has quickly become a “star product” in treating hematological malignancies.

CAR-T cells, as the name implies, are T cells expressing CAR molecules. The CAR molecule comprises a transmembrane domain, an antigen recognition domain, and a signal transduction domain. The first generation CAR molecule contains an intracellular T cell activation domain of CD3ζ. The subsequent generations of CAR molecules added more costimulatory domains, such as CD28, 4-1BB, etc., to enhance CAR-T cells’ vitality and killing ability (check more on Article 1 from the series “Decoding Cell and Gene Therapy”).

Traditional CAR-T cell construction method

Traditionally, CAR-T cells are produced by transfection with viral vectors. In this process, the CAR sequence is delivered by the lentivirus and integrated into the target genome. This method can ensure the adequacy of integration and at the same time enable the CAR construct to be stably expressed.

However, using viral vectors to construct CAR-T cells can cause several problems related to the random integration of CAR sequences, such as introducing insertion mutations, causing heterogeneous expression of CAR or silencing of CAR expression.

A new method for targeted CAR insertion using CRISPR

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Continuous breakthroughs in gene editing tools have led to the continuous and rapid development of the global biomedical industry. At present, CRISPR technology has been used in the construction of CAR-T cells by disrupting endogenous genes.

Unlike random integration virus synthesis methods, the CRISPR/Cas9 system can target CAR to specific genes of interest to achieve a precise gene integration.

In the CRISPR editing method, the guide RNA can recognize a specific DNA sequence through its crRNA components, a sequence of about 20 nucleotides, which can be annealed in the presence of tracrRNA to form a ribonucleoprotein complex with Cas9. Under the catalysis of the related Cas9 endonuclease, the CRISPR complex produces double-strand breaks at targeted genomic sites. Subsequently, cell repair mechanisms, such as non-homologous end joining and homologous-directed recombination, are activated to repair the sequence.

In the production of CAR-T cells, homologous directed recombination can be used to achieve targeted gene insertion of large DNA sequences. For example, Eyquem et al. used CRISPR/Cas9 tools, Cas9 mRNA and gRNA, to successfully insert a specific CAR targeting CD-19 into the TRAC site encoding the alpha chain of the T cell receptor [1].

Recently, Zhang et al. used CRISPR/Cas9 tools to develop CAR-T cells targeting two genomic loci AAVS1 and PD1 [2].

They targeted the non-viral CAR DNA template (including CD19 antigen recognition domain and 4-1BB-CD3ζ signal domain) to the AAV1 site through CRISPR/Cas9. The generated CAR-T cells were comparable to those conventionally produced by lentivirus. T cells have similar cell expansion and tumor-killing properties.

In addition, compared with CAR-T cells generated by lentivirus, targeting the same CAR molecule to the PD1 site through CRISPR/Cas9 can make CAR-T cells have better amplification characteristics and be more effective in inhibiting the growth of tumors.

Subsequently, the researchers conducted a clinical trial to test the safety and effectiveness of CAR-T cells targeted by CRISPR/Cas9 PD1 in patients with relapsed and refractory B-cell non-Hodgkin lymphoma. For example, Zhang et al. used ribonucleoprotein complexes and a non-viral CAR DNA template to electroporate patient-derived T cells. The results of this clinical trial showed that the CAR-T cells synthesized by this method caused no serious adverse events and successfully achieved complete remission in two representative patients.

In general, the specificity of the CRISPR/Cas gene editing system is rapidly being used to develop and research autologous and allogeneic CAR-T cells. The use of ribonucleoprotein (RNP) formed by Cas9 and sgRNA to replace plasmids and lentiviruses is fast and safe, has lower off-target effects, and has higher editing efficiency. It has gradually become a more efficient way to achieve CRISPR/Cas9 technology. Relying on this new non-viral knock-in strategy for CAR-T cell synthesis can ensure safe and effective cell therapy products.

P.S. In the study Zhang et al. successfully developed non-viral genome specific integrated CAR-T cells for preclinical and clinical research using sgRNA synthesized by GenScript. The sgRNA synthesized by GenScript helps researchers build CAR-T cells based on the CRISPR method. The CAR-T cells constructed by this method have better expansion characteristics and can inhibit the growth of tumor cells more effectively. GenScript has now launched EasyEdit sgRNA synthesis service with low toxicity, more stability and high editing efficiency.

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[1] Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature (2017) doi:10.1038/nature21405.

[2] Zhang, J. et al. Development and clinical evaluation of non-viral genome specific targeted CAR T cells in relapsed/refractory B-cell non-Hodgkin lymphoma. medRxiv 2020.09.22.20199786; doi:

[3] GenScript, “Novel CRISPR-based Non-viral Approach for CAR T Cell Generation”, achieved at