citations

                     Single-stranded HDR templates with truncated Cas12a binding sequences 
improve knock-in efficiencies in primary human T cells
                

Authors:

Ana-Maria Nitulescu (1,2), Weijie Du (1,2,#), Viktor Glaser (1,2), Jonas Kath (1,2), Robert Greensmith (3,4), Nanna Steengaard Mikkelsen (5), Maik Stein (1,2), Rasmus O. Bak (5), Michael Kaminski (2,3,4), Dimitrios L. Wagner (1,2,6,*).

In:

Source:
Publication Date: (2024)
Issue: :

Research Area:

Cancer Research/Cell Biology

Immunotherapy / Hematology

Basic Research

Cells used in publication:

T cell, human stim.: Species: human; Tissue Origin: blood

Platform:

4D-Nucleofector® X-Unit

Experiment

Electroporation
Non-viral knock-ins in T cells were performed as recently described (27). 48 hour-stimulated primary T cells were harvested, counted and washed twice in sterile PBS for 10 minutes, first at 150 x g and then at 100 x g. Afterwards, 1 x 10^6 cells were resuspended in 20 µL P3-buffer (Lonza) per electroporation reaction and added to the RNP-HDRT mix. The suspension was carefully transferred to a 16-strip electroporation cuvette, which was then tapped on the bench repeatedly to guarantee proper positioning of fluids within the strip and the absence of bubbles that would interfere with the electric current. The cells were electroporated with the 4D-Nucleofector device (Lonza) using the program EH-115 according to previous reports (22, 27). Quickly after, 90 µL of pre-warmed medium were added per electroporation reaction, and the strip was incubated for 10 minutes at 37°C. Lastly, the cells were seeded to a 96-well round-bottom plate (50 µL cells/well) at a density of 0.5 x 10^6 cells per well.

Abstract

Non-viral gene editing via CRISPR-Cas12a offers an alternative to Cas9-based methods, providing better targeting of AT-rich regions, simplified guide RNA manufacturing, and high specificity. However, the efficacy of editing outcomes is subject to various factors, with template format playing a crucial role. Currently, the predominant non-viral template format for inducing homology-directed repair (HDR) after nuclease-induced DNA breaks is doublestranded DNA (dsDNA), which is toxic when transfected at high doses. Previous studies have demonstrated that using single-stranded DNA (ssDNA) with flanking double-stranded Cas-target-sequences (CTS) as a repair template for Cas9-mediated gene editing can mitigate this toxicity and increase knock-in efficiency. Here, we investigate CTS design for AsCas12a Ultra by exploring PAM orientation and binding requirements of the Cas12acrRNA complex. Additionally, we rule out in vitro ssDNase activity of AsCas12a Ultra under cell-physiological Mg2+ conditions. Finally, we showcase the advantage of using ssDNA with double-stranded CTS end modifications (ssCTS) at high doses for delivering clinically relevant transgenes of varying sizes into three T-cell receptor-CD3 complex genes (TRAC, CD3?, CD3e), achieving up to 90% knock-in rates for a 0.8kb insert at the CD3e locus. Overall, AsCas12a Ultra and ssCTS donors represent a platform for highly efficient knock-in in primary human T cells with minimal toxicity.

Single-stranded HDR templates with truncated Cas12a binding sequences improve knock-in efficiencies in primary human T cells