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PAM-flexible genome editing with an engineered chimeric Cas9.

Lin ZhaoSabrina R T KosekiRachel A SilversteinNadia AmraniChristina PengChristian KrammeNatasha SavicMartin PacesaTomas C RodriguezTeodora StanEmma TysingerLauren HongVivian YudistyraManvitha R PonnapatiJoseph M JacobsonGeorge M ChurchNoah JakimoRay TruantMartin JinekBenjamin P KleinstiverErik J SontheimerPranam Chatterjee
Published in: Nature communications (2023)
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.
Keyphrases
  • crispr cas
  • genome editing
  • cell therapy
  • genome wide
  • amino acid
  • dna methylation
  • bone marrow
  • cancer therapy
  • protein protein
  • mesenchymal stem cells