| Keywords [eng] |
CRISPR-Cas, CRISPR, genome editing, epigenome editing, methylation sensitivity, directed evolution, Cas9, DNA methylation, protein engineering |
| Abstract [eng] |
CRISPR (clustered regularly interspaced short palindromic repeats) locus and Cas (CRISPR-associated) proteins provide adaptive immunity in bacteria and archaea by integrating foreign DNA into the CRISPR locus. This sequence is transcribed into a guide RNA (gRNA), which directs a Cas nuclease to recognize and cleave target DNA adjacent to a PAM (protospacer adjacent motif). The gRNA can be programmed to target any sequence of interest, enabling precise genome editing and diagnostic applications. Epigenetic modifications can influence CRISPR-Cas activity. Though target or PAM methylation does not hinder the widely used Streptococcus pyogenes Cas9, DNA condensation in highly methylated region can, making certain genomic sites less accessible for editing. In contrast, Acidothermus cellulolyticus Cas9 (AceCas9) senses methylation. Its cleavage activity depends on whether the first (but not the second) cytosine of its PAM sequence is methylated (5′-NNNCC-3′). This property offers potential for methylation-sensitive gene editing. AceCas9 operates in Escherichia coli and Clostridium thermocellum, but function in human cells remains unknown. This work aimed to determine this. This study demonstrated that AceCas9 can be expressed in human embryonic kidney (HEK293T) cells and enter the nucleus, which are key processes preceding genome editing. To assess editing in human cells, two endogenous human gene loci, PCSK9 and UBE3A, were targeted with AceCas9. AceCas9-mediated editing in human cells remains inconclusive due to low levels of detected editing and the lack of clear distinction in sequencing data. This study also aimed to evolve AceCas9 to recognize a 5′-NNNCG-3′ PAM, as cytosine methylation in CpG dinucleotides is the most prevalent DNA methylation in human cells. A library of 11600 AceCas9 PAM-interacting domain variants was constructed and a ccdB-based directed evolution method was employed, linking Cas9 activity with bacterial survival. However, none of the tested variants were able to recognize a target with the 5′-NNNCG-3′ PAM. These findings suggest that while AceCas9 can be expressed in human cells and localized to the nucleus, it requires further optimization for genome editing in human cells. The inability to evolve AceCas9 to recognize a 5′-NNNCG-3′ highlights our need for continued development of human-specific genome editing tools. |
