Data from: Orthogonal transcriptional modulation and gene editing using multiple CRISPR/Cas systems

CRISPR/Cas-based transcriptional activation (CRISPRa) and interference
(CRISPRi) enable transient programmable gene regulation by recruitment or
fusion of transcriptional regulators to nuclease-deficient Cas (dCas).
Here we expand on the emerging area of transcriptional engineering and RNA
delivery by benchmarking combinations of RNA-delivered dCas and
transcriptional modulators. We utilize dCas9 from Staphylococcus aureus
and Streptococcus pyogenes for orthogonal transcriptional modulation to
upregulate one set of genes while downregulating another. We also
establish trimodal genetic engineering by combining orthogonal
transcriptional regulation with gene knockout by Cas12a (Acidaminococcus;
AsCas12a) ribonucleoprotein (RNP) delivery. To simplify trimodal
engineering, we explore optimal parameters for implementing truncated
sgRNAs to make use of SpCas9 for knockout and CRISPRa. We find the Cas9
protein:sgRNA ratio to be crucial for avoiding sgRNA cross-complexation
and for balancing knockout and activation efficiencies. We demonstrate
high efficiencies of trimodal genetic engineering in primary human T cells
while preserving basic T cell health and functionality. This study
highlights the versatility and potential of complex genetic engineering
using multiple CRISPR/Cas systems in a simple, one-step process yielding
transient transcriptome modulation and permanent DNA changes. We believe
such elaborate engineering can be implemented in regenerative medicine and
therapies to facilitate more sophisticated treatments.