Paper of the month August 2019

Improving CRISPR-Cas9 Genome Editing Efficiency by Fusion with Chromatin-Modulating Peptides

During the last years CRISPR-Cas9 has become a common tool for genome editing. However, the editing efficiency is often limited and depends heavily on the genomic site. In this paper the authors show that fusing Cas9 with chromatin modulating peptides (CMPs) increases Cas9 activity by up to several fold. This strategy was termed "CRISPR-chrom".

In a first step the authors created 33 fusion proteins, in which one CMP was fused to either end of the commonly used Streptococcus pyogenes Cas9 (SpyCas9). Editing efficiency was tested and compared to unmodified SpyCas9, resulting in a selection of several candidate CMPs with increased activity. Subsequently SpyCas9 was fused with two of the candidate CMPs, one at either end, in different combinations. Testing and comparing editing efficiency to unmodified SpyCas9 resulted in three top candidate fusion proteins with increased activity. At one end all selected fusion proteins contain HMGN1, which belongs to a group of proteins that have been linked to chromatin decompaction. Additionally, one of three CMPs with native DNA binding activity (named HMGB1 box A, histone H1 central globular domain or CHD1 DNA binding domain) was fused to the other end of SpyCas9.

Editing efficiency of those three top candidate fusion proteins was tested on additional genomic sites. All three fusion proteins could reach higher editing efficiencies for all tested refractory target sites and many of the tested moderately to highly active target sites. The fusion proteins were further tested for their effects on homology-directed repair (HDR) efficiency. During HDR an additional repair template is provided that can be used to introduce precise alterations in the genome. HDR efficiency was higher for all three fusion proteins.

While SpyCas9 is widely used for genome editing, alternative Cas9 variants can increase the targeting density in the genome. However, alternative Cas9 variants often show highly variable editing efficiency, depending on the genomic site. The CRISPR-chrom strategy was thus tested for two previously described Cas9 variants (derived from Streptococcus pasteurianus or Campylobacter jejuni) as well as 4 previously uncharacterized Cas9 variants (derived from Bacillus smithii, Lactobacillus rhamnosus, Mycoplasma canis or Parasutterella excrementihominis). For all tested Cas9 variants fusions with CMPs could increase editing efficiency for almost all targets compared to unmodified Cas9 variants. This demonstrates that the CRISPR-chrom strategy is also useful for alternative Cas9 variants.

Off-target effects describe editing at unintended targets. To test whether the fusion proteins show increased off-target activity compared to nonmodified Cas9 variants, 72 predicted off-targets have been analyzed. Except for one target-site, no higher off-target activity was visible for fusion proteins.

To summarize the results, the authors show that Cas9 fusion with CMPs increases genome editing efficiency for many target sites, without increasing off-target activity (with one exception). The CRISPR-chrom strategy can be applied to different Cas9 variants.