Skip to main content
Premium Trial:

Request an Annual Quote

New CRISPR-Cas9 Variants Improve Targeting Ability of Base Editors

NEW YORK (GenomeWeb) – A team led by researchers at the Broad Institute and the University of California, Berkeley has engineered new CRISPR-Cas9 variants that widen the targeting scope of base editors, thus broadening the number of human pathogenic variants that could potentially targeted.

"Indeed, an analysis of human pathogenic single nucleotide polymorphisms in ClinVar reflects a substantial improvement in the fraction of targetable SNPs when considering the expanded [cytosine or adenine base editors'] editing windows compared with their unpermuted counterparts," the authors wrote in their study in Nature Biotechnology today

For base editing to successfully occur, the target sequence must be near a protospacer adjacent motif (PAM) that is recognized by the Cas9 domain, and the target nucleotide must be located within the editing window of the base editor. To increase the targeting scope of base editors, the researchers engineered six optimized variants of the adenine base editor (ABE) ABEmax, which use Streptococcus pyogenes Cas9 (SpCas9) variants compatible with non-NGG PAMs.

They also used circularly permuted Cas9 variants (CP-Cas9) to produce four cytosine base editors (CBEs) and four ABEs with an editing window of up to about eight to nine nucleotides, compared to their original editing window of about four to five nucleotides.

"The resulting CP-CBEmax variants exhibit higher product purities, in addition to expanded editing windows, while CP-ABEmax variants maintain the high product purities typical of ABEs," the authors wrote. "These CBE and ABE variants expand the targeting scope of base editing."

The researchers began by creating ABEmax variants that replaced the SpCas9 nickase component with two engineered SpCas9 variants with altered PAM specificities: VRQR-SpCas9 targeting the NGA PAM sequence and VRER-SpCas9 targeting the NGCG PAM sequence. They named these editors VRQR-ABEmax and VRER-ABEmax. They then evaluated the base-editing activity of these ABE variants at six endogenous human genomic loci for each PAM in human HEK293T cells and found that although editing with ABEmax across six endogenous NGA PAM-containing sites resulted in low editing efficiency, editing with VRQR-ABEmax resulted in a 3.2-fold average improvement across all six sites.

They then tested the editing efficiency of ABEmax at six endogenous genomic sites in HEK293T cells containing NGCG PAMs and again observed minimal activity. In contrast, editing with VRER-ABEmax at those sites resulted in a sevenfold improvement over ABEmax.

Further experiments showed that the VRQR-, VRER- and SpCas9-NG variants were compatible with the ABEmax architecture and retained base-editing activity at sites containing their cognate nonNGG PAMs.

To expand the targeting scope of ABE even further, the researchers tried to examine whether Staphylococcus aureus Cas9 (SaCas9) could also be compatible with the ABEmax architecture. SaCas9 naturally targets NNGRRT PAMs, and an evolved variant called SaKKH recognizes NNNRRT PAMs.

The team generated both SaCas9 and SaKKH-ABEmax variants and tested them on six endogenous NNGRRT PAM sites and six endogenous NNHRRT PAM sites in HEK293T cells. They observed moderate editing efficiencies for SaABEmax and SaKKHABEmax, which contrasted with the high activities of SaCas9-derived CBEs that generally edit more efficiently than the corresponding SpCas9 CBE.

"These results suggest that further engineering or evolution may benefit targeting ABE with SaCas9 derivatives," the authors noted.

Given the potential utility of base editors with shifted or expanded activity windows, the researchers next sought to engineer base editor architectures that enabled editing at different protospacer positions. They hypothesized that circularly permuted Cas9 variants might result in expanded or otherwise altered activity windows, and chose five SpCas9 circular permutants — CP1012, CP1028, CP1041, CP1249, and CP1300 — based on both retention of DNA binding activity and predicted proximity to the single-strand DNA loop. They generated five CP-CBEmax and five CP-ABEmax variants and transfected them into HEK293T cells to test their base-editing activity at five endogenous genomic sites containing adenines and cytosines throughout the target 20-nucleotide protospacer.

The researchers found that four of the five CP-CBE variants were capable of base editing at all five sites without substantial indel formation, while CP1300-CBEmax demonstrated highly site-dependent base-editing activity. Three of the top four CP-CBEmax variants exhibited efficient editing activity, and CP1012-CBEmax and CP1028-CBEmax in particular showed broadening of the editing window from the canonical positions four to eight to positions four to 11 of the protospacer.

Similarly, most of the CP-ABEmax variants also exhibited a broadening of the editing window, the researchers said. CP-ABEmax variants retained efficient editing activity similar to that of ABEmax, and both ABEmax and the CP variants generated minimal indels.

Significantly, the researchers found that the window-broadening effect of the CP-ABEmax variants was pronounced, generally resulting in an expansion from the canonical window of protospacer positions four to seven for ABEmax to a window spanning positions four to 12.

When they measured the off-target base-editing efficiency of the CP base editors, the researchers found that it was similar to or less than that of CBEmax or ABEmax for C or A nucleotides within the canonical editing window. For C or A nucleotides outside of the canonical editing window, the expanded editing windows of the CP base editors resulted in higher off-target editing than CBEmax or ABEmax, in some cases.

"Together, these results demonstrate that circularly permuting the Cas9 nickase domain of base editors results in CBEmax and ABEmax variants with broadened or shifted editing windows," the authors concluded.