58. Engineered Cas9 Variants with Novel PAM Specificities Expand the Targeting Range of CRISPR/Cas Nucleases
Year: 2015
Type: article
Abstract: Specificity and targeting range are important considerations when selecting an engineered nuclease platform for functional studies or therapeutic applications. The use of CRISPR/Cas9 to edit virtually any genome has enabled researchers to rapidly knock out genes or to introduce precise genomic changes. A number of studies have explored and improved the specificity of the CRISPR/Cas9 platform but the targeting range remains limited to certain sequences. The most widely used Cas9 from Streptococcus pyogenes (SpCas9) recognizes a protospacer adjacent motif (PAM) of the form NGG, a constraint that restricts the range of sequences that can be edited. Combined with the 5’ nucleotide requirement for guide-RNA expression from a polIII promoter, this restriction can be limiting particularly when one is using CRISPR/Cas9 nucleases to introduce precise mutations by homology-directed repair (HDR), which requires that double-strand breaks be introduced very closely to the site of sequence alteration. To address this challenge, we developed a heterologous genetic system that enabled us to rapidly screen libraries of Cas9 clones and identify variants with distinct cleavage specificities. Using this technology, we identified and optimized two different Cas9 variants that recognize novel PAMs. We demonstrate that these variants enable targeting of sites with non-canonical PAM sequences that were previously inaccessible with wild-type SpCas9, more than doubling the total range of sequences that can be edited by HDR and by error-prone non-homologous end-joining (NHEJ). We demonstrate that these variants can robustly modify endogenous genes in human cells and that they are compatible with previously described specificity improvements to SpCas9 such as truncated gRNAs. Furthermore, we also show Cas9 nucleases from other bacterial species such as Staphylococcus aureus and Streptococcus thermophilus can also be assessed for activity in our heterologous genetic system, suggesting that these alternative Cas9s might also be modified in their PAM specificities using the same approach we undertook with SpCas9. These orthogonal Cas9 nucleases offer the advantage of being substantially smaller in size than SpCas9, an important factor when considering packaging limits for viral delivery of CRISPR/Cas9 components. Taken together, our results more than double the targeting range of CRISPR/Cas9 nuclease platform and improve the prospects for translation of these reagents to clinical settings.
Source: Molecular Therapy
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,500
OpenAlex ID: W2561834051