RESUMEN
CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN (R = A or G, Y = C or T) PAM preference, with the N-terminus of Sc + +, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse PAMs and disease-related loci for potential therapeutic applications. In total, the approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Sistemas CRISPR-Cas/genética , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , GenomaRESUMEN
CRISPR-associated (Cas) DNA-endonucleases are remarkably effective tools for genome engineering, but have limited target ranges due to their protospacer adjacent motif (PAM) requirements. We demonstrate a critical expansion of the targetable sequence space for a type II-A CRISPR-associated enzyme through identification of the natural 5[Formula: see text]-NAAN-3[Formula: see text] PAM preference of Streptococcus macacae Cas9 (SmacCas9). To achieve efficient editing activity, we graft the PAM-interacting domain of SmacCas9 to its well-established ortholog from Streptococcus pyogenes (SpyCas9), and further engineer an increased efficiency variant (iSpyMac) for robust genome editing activity. We establish that our hybrids can target all adenine dinucleotide PAM sequences and possess robust and accurate editing capabilities in human cells.
Asunto(s)
Adenina/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Fosfatos de Dinucleósidos/metabolismo , Motivos de Nucleótidos/genética , Secuencia de Aminoácidos , Proteína 9 Asociada a CRISPR/química , Edición Génica , Células HEK293 , Humanos , Reproducibilidad de los Resultados , Streptococcus/genéticaRESUMEN
CRISPR enzymes require a protospacer-adjacent motif (PAM) near the target cleavage site, constraining the sequences accessible for editing. In the present study, we combine protein motifs from several orthologs to engineer two variants of Streptococcus canis Cas9-Sc++ and a higher-fidelity mutant HiFi-Sc++-that have simultaneously broad 5'-NNG-3' PAM compatibility, robust DNA-cleavage activity and minimal off-target activity. Sc++ and HiFi-Sc++ extend the use of CRISPR editing for diverse applications.
Asunto(s)
Secuencias de Aminoácidos/genética , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , División del ADN , Edición Génica/métodos , Streptococcus/genéticaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.