citations

Engineering of CRISPR-Cas12b for human genome editing.

Authors:

Strecker J, Jones S, Koopal B, Schmid-Burgk J, Zetsche B, Gao L, Makarova KS, Koonin EV, Zhang F.

In:

Source: Nature
Publication Date: (2019)
Issue: 10(1): 212

Research Area:

Immunotherapy / Hematology

Cells used in publication:

T cell, human stim.: Species: human; Tissue Origin: blood
CD4+, human: Species: human; Tissue Origin: blood

Platform:

4D-Nucleofector® X-Unit

Experiment

Cells were electroporated using the Amaxa P3 Primary Cell 4D-Nucleofector X Kit (Lonza). Per reaction, 3 × 105 stimulated CD4+ T cells were pelleted and resuspended in 20 µL of P3 buffer. In total, 4.5 µM Cas9 or Cas12b protein, precomplexed with crRNA and tracrRNA, was added and the mixture transferred to the electroporation cuvette. Cells were electroporated using program EH-115 on
the Amaxa 4D-Nucleofector (Lonza). Eighty microliters of prewarmed complete media was immediately added to the cells post pulse and the cells were incubated at 37 °C for 30 min to recover in the cuvette. After recovery, 50 µL of the cell suspension was added to 50 µL of complete medium plus 80 IU/mL IL-2 (STEMCELL Technologies) for a final concentration of 40 IU/mL IL-2.

Abstract

The type-V CRISPR effector Cas12b (formerly known as C2c1) has been challenging to develop for genome editing in human cells, at least in part due to the high temperature requirement of the characterized family members. Here we explore the diversity of the Cas12b family and identify a promising candidate for human gene editing from Bacillus hisashii, BhCas12b. However, at 37?°C, wild-type BhCas12b preferentially nicks the non-target DNA strand instead of forming a double strand break, leading to lower editing efficiency. Using a combination of approaches, we identify gain-of-function mutations for BhCas12b that overcome this limitation. Mutant BhCas12b facilitates robust genome editing in human cell lines and ex vivo in primary human T cells, and exhibits greater specificity compared to S. pyogenes Cas9. This work establishes a third RNA-guided nuclease platform, in addition to Cas9 and Cpf1/Cas12a, for genome editing in human cells.

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