A Cas9 with PAM recognition for adenine dinucleotides

• DOI: 10.1038/s41467-020-16117-8
• Publication Date: 2020-05-18T10:02:50Z
• Authors: Pranam Chatterjee; Jooyoung Lee; Lisa Nip; Sabrina R. T. Koseki; Emma Tysinger; Erik J. Sontheimer; Joseph M. Jacobson; Noah Jakimo
• Keywords: CRISPR-Cas9 genome editing; 0301 basic medicine; Bioinformatics; Science; Article; 03 medical and health sciences; Nucleic Acids; CRISPR-Associated Protein 9; Enzymes and Coenzymes; Humans; Amino Acid Sequence; Nucleotide Motifs; Synthetic biology; Gene Editing; Nucleotides; Adenine; Q; Computational Biology; Reproducibility of Results; Streptococcus; Genetics and Genomics; and Nucleosides; Computational biology and bioinformatics; 3. Good health; HEK293 Cells; Genetic Phenomena; Dinucleoside Phosphates

Abstract 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 type II-A enzyme through identification natural 5 $$^{\prime}$$ <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msup><mml:mrow/><mml:mrow><mml:mo>′</mml:mo></mml:mrow></mml:msup></mml:math> -NAAN-3 PAM preference Streptococcus macacae Cas9 (SmacCas9). To achieve efficient editing activity, we graft PAM-interacting domain SmacCas9 its well-established ortholog from pyogenes (SpyCas9), and further engineer an increased efficiency variant (iSpyMac) robust activity. establish that our hybrids can all adenine dinucleotide sequences possess accurate capabilities in human cells.