The molecular basis for recognition of 5′-NNNCC-3′ PAM and its methylation state by Acidothermus cellulolyticus Cas9

Das, Anuska; Hand, Travis H.; Smith, Chardasia L.; Wickline, Ethan; Zawrotny, Michael; Li, Hong

doi:10.1038/s41467-020-20204-1

Cited by 20 publications

(21 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the structure of AceCas9 ( 44 ), whose PAM preference is NNNCC, the fourth C forms a hydrogen bond with Glu1044, which is positioned by Arg1091, and the paired G forms a hydrogen bond with Arg1088. The fifth C’s paired G on the TS forms bidentate hydrogen bonds with Arg1091 ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Decoding CRISPR–Cas9 PAM recognition with UniDesign

Huang

Zhou

Yang

et al. 2023

Preprint

View full text Add to dashboard Cite

The critical first step in CRISPR-Cas mediated gene editing is recognizing a preferred protospacer adjacent motif (PAM) on target DNAs by the protein's PAM-interacting amino acids (PIAAs). Thus, accurate computational modeling of PAM recognition is useful in assisting CRISPR-Cas engineering to relax or tighten PAM requirement for subsequence applications. Here we describe a universal computational protein design framework (UniDesign) for designing protein-nucleic acid interactions. As a proof of concept, we applied UniDesign to decode the PAM-PIAA interactions for eight Cas9 proteins. We show that, given native PIAAs, the UniDesign-predicted PAMs are largely identical to the natural PAMs of all Cas9s. In turn, given natural PAMs, the computationally redesigned PIAA residues largely recapitulated the native PIAAs (>70% and >80% in terms of identity and similarity, respectively). These results demonstrate that UniDesign faithfully captures the mutual preference between natural PAMs and native PIAAs, suggesting it as a useful tool for engineering CRISPR-Cas and other nucleic acid-interacting proteins. UniDesign is open-sourced at https://github.com/tommyhuangthu/UniDesign.

show abstract

Section: Resultsmentioning

confidence: 99%

Decoding CRISPR–Cas9 PAM recognition with UniDesign

Huang

Zhou

Yang

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…AceCas9 was cloned in pET28b vector and expressed in Escherichia coli BL21 strain, and was purified as previously described 16,20 . Briefly, the protein was purified by Nickel affinity chromatography followed by heparin ion exchange.…”

Section: Protein and Nucleic Acid Sample Preparationmentioning

confidence: 99%

“…Though the wild-type AceCas9 has slower rate of cleavage than that of SpyCas9, protein directed engineering has resulted in a catalytic enhanced AceCas9 that has a comparable cleavage rate to that of SpyCas9 9 . Importantly, AceCas9 is uniquely sensitive to DNA methylation and does not cleave DNA with a 5’-NNN m CC-3’ PAM 16 . Both the unique and the conserved biochemical properties of AceCas9 make it an excellent candidate as a biotechnology tool and a model system for studying genome editing enzymes.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Conformational Changes Regulate Metal Coordination in the Catalytic Sites of Cas9

Das

Rai

Roth

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

The control of CRISPR-Cas9 activities plays an essential role in its safe adoption for clinical and research applications. Previous studies have identified conformational dynamics of Cas9 as a key regulatory element for its enzymatic activity. The molecular basis for how conformations influence the catalytic processes at both nuclease domains, however, remains elusive. Here we present well-resolved cryo-EM structures of the active Acidothermus cellulolyticus Cas9 (AceCas9) complexes representing pre-cleavage (2.9 &Aring), two reaction intermediate (2.2 &Aring and 2.4 &Aring) and two post-cleavage (2.6 &Aring and 2.7 &Aring) states that reveal active site rearrangement during DNA binding and phosphodiester bond breakage by Cas9. Strikingly, large domain rearrangements in Cas9 triggered by the cognate DNA result in subtle changes in active sites that facilitate coordination of metal ions required for catalysis. The two reaction intermediate structures further reveal small oscillations in domain conformations, which alternates the reactive states of the two catalytic centers, thereby coupling cleavage of the two DNA strands. Consistent with the roles of conformations in organizing the active sites, adjustments of metal coordination ligands lead to altered metal specificity.

show abstract

“…We first applied the protein directed evolution method to a Type II-C Cas9 from Acidothermus cellulolyticus (AceCas9). Due to its thermostability and recently discovered sensitivity to DNA methylation, 36,37 AceCas9 has been used for gene editing in model thermophilic bacteria for biofuel processing 38 and is anticipated to have expanded application in epigenetics. AceCas9 was previously shown to be most efficient in cleaving protospacers with 24 nt of spacer complementarity.…”

mentioning

confidence: 99%

Catalytically Enhanced Cas9 Through Directed Protein Evolution

et al. 2021

Self Cite

View full text Add to dashboard Cite

Guided by the extensive knowledge of CRISPR-Cas9 molecular mechanisms, protein engineering can be an effective method in improving CRISPR-Cas9 toward desired traits different from those of their natural forms. Here, we describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9) by targeting a shortened protospacer within a toxic gene. We demonstrate the effectiveness of this method with a previously characterized Type II-C Cas9 from Acidothermus cellulolyticus (AceCas9) and show by enzyme kinetics an up to fourfold improvement of the in vitro catalytic efficiency by AceCECas9. We further evolved the more widely used Streptococcus pyogenes Cas9 (SpyCas9) and demonstrated a noticeable improvement in the SpyCECas9-facilitated homology directed repair-based gene insertion in human colon cancer cells.

show abstract

The molecular basis for recognition of 5′-NNNCC-3′ PAM and its methylation state by Acidothermus cellulolyticus Cas9

Cited by 20 publications

References 52 publications

Decoding CRISPR–Cas9 PAM recognition with UniDesign

Decoding CRISPR–Cas9 PAM recognition with UniDesign

Conformational Changes Regulate Metal Coordination in the Catalytic Sites of Cas9

Catalytically Enhanced Cas9 Through Directed Protein Evolution

Contact Info

Product

Resources

About