Processing
Background
Cas9 genome editing, while valuable for many research purposes, is restricted to genomic sites containing a Cas9-recognizable protospacer-adjacent motif (PAM). GC-rich regions are more amenable to Cas9 genome editing than are AT-rich regions. Cas12a recognizes TTTV PAM sequences and thus is frequently the CRISPR-Cas endonuclease of choice for editing in AT-rich regions. Wild-type (WT) Cas12a also compares favorably to WT Cas9 in terms of site specificity. However, WT Cas12a has been shown to have significantly less potency than wild-type Cas9. Zhang et al. set about to develop AsCas12a Ultra, a modification of A.s. Cas12a that would maintain site specificity while demonstrating high levels of editing activity.
Experiment and results
As described in a previous DECODED article, a process of selecting and enriching for A.s. Cas12a mutations with increased cleavage efficiency was performed in E. coli. Multiple rounds of selection and enrichment for higher-activity Cas12a mutants resulted in isolation of M537R/F870L (AsCas12a Ultra). Then, using Spec/SEAM-seq, the researchers found that AsCas12a Ultra had binding and cleavage specificities similar to WT Cas12a. As described in the aforementioned DECODED article, AsCas12a Ultra also showed greatly improved potency over WT Cas12a, including in low temperature conditions (30°C).
Using a variety of primary cell types (T cells, HSCs, NK cells, and iPSCs), the researchers employed electroporation of ribonucleoproteins (RNPs) consisting of CRISPR RNAs (crRNAs) combined with either WT or AsCas12a Ultra to edit many different genomic target loci. Concentration-response curves demonstrated an increase in editing efficiency with AsCas12a Ultra. Editing efficiencies of WT and AsCas12a Ultra were investigated by next generation sequencing (NGS) and demonstrated to be equivalent.
The researchers investigated an approach to develop allogenic T cells by triple knockout of TRAC, B2M, and CIITA using AsCas12a Ultra. Greater than 90% editing efficiency was observed at all three genomic sites. In a follow-up experiment, a donor template carried by AAV6 was introduced to allow expression of a transgene. Genes for green fluorescent protein (GFP) and mCherry were successfully introduced at close to 60% knock-in rates—even double knock-in of these two fluorescent reporters was achieved at a greater than 20% rate.
Finally, the researchers studied the ability of AsCas12a Ultra to generate allogenic chimeric antigen receptor (CAR) NK cells. First, they used AsCas12a Ultra in NK cells to knock out the TGFBR2 gene, which codes for one subunit of the TGFb receptor. They showed that in response to TGFb, the edited NK cells more effectively killed SK-OV-3 ovarian tumor spheroids than unedited NK cells while also exhibiting lower levels of SMAD2/3 phosphorylation, explaining the mechanism of this cytotoxic effect. Then, using AAV6, the researchers knocked in a CAR which targets the epidermal growth factor receptor. The resultant knock-in cells (αEGFR-CAR+ NK) were then co-cultured with EGFR+ PC-3 prostate tumor spheroids, leading to decreased spheroid size. The data demonstrated that the cytotoxicity of NK cells was increased by these genome editing modifications.
