Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules

Shams, Arik; Higgins, Sean; Fellmann, Christof; Laughlin, Thomas G.; Oakes, Benjamin L.; Lew, Rachel J.; Lukarska, Maria; Arnold, Madeline; Staahl, Brett T.; Doudna, Jennifer A.; Savage, David F.

doi:10.1101/2020.10.19.344077

Cited by 6 publications

(5 citation statements)

References 68 publications

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“…Deletion of the entire BH severely reduced the catalytic activity. Similarly, a BH deletion variant of SpyCas9 was inactive ( 58 ). This shows that the BH is an essential segment for both enzymes.…”

Section: Discussionmentioning

confidence: 99%

Decoupling the bridge helix of Cas12a results in a reduced trimming activity, increased mismatch sensitivity and impaired conformational transitions

Wörle

Jakob

Schmidbauer

et al. 2021

Nucleic Acids Research

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The widespread and versatile prokaryotic CRISPR–Cas systems (clustered regularly interspaced short palindromic repeats and associated Cas proteins) constitute powerful weapons against foreign nucleic acids. Recently, the single-effector nuclease Cas12a that belongs to the type V CRISPR–Cas system was added to the Cas enzymes repertoire employed for gene editing purposes. Cas12a is a bilobal enzyme composed of the REC and Nuc lobe connected by the wedge, REC1 domain and bridge helix (BH). We generated BH variants and integrated biochemical and single-molecule FRET (smFRET) studies to elucidate the role of the BH for the enzymatic activity and conformational flexibility of Francisella novicida Cas12a. We demonstrate that the BH impacts the trimming activity and mismatch sensitivity of Cas12a resulting in Cas12a variants with improved cleavage accuracy. smFRET measurements reveal the hitherto unknown open and closed state of apo Cas12a. BH variants preferentially adopt the open state. Transition to the closed state of the Cas12a-crRNA complex is inefficient in BH variants but the semi-closed state of the ternary complex can be adopted even if the BH is deleted in its entirety. Taken together, these insights reveal that the BH is a structural element that influences the catalytic activity and impacts conformational transitions of FnCas12a.

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Section: Discussionmentioning

confidence: 99%

Decoupling the bridge helix of Cas12a results in a reduced trimming activity, increased mismatch sensitivity and impaired conformational transitions

Wörle

Jakob

Schmidbauer

et al. 2021

Nucleic Acids Research

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“…In the past, DMS has been applied, for instance, to characterize human disease mutations (2), dissect protein-coding regions in the adeno-associated virus genome (3) and investigate the mutational fitness landscape of CRISPR-Cas9 (4,5). Moreover, comprehensive DMS datasets comprising protein single and double mutants were used for detailed analysis of intramolecular interactions, based on which the three-dimensional protein structure could be inferred (6).…”

Section: Introductionmentioning

confidence: 99%

A deep mutational scanning platform to characterize the fitness landscape of anti-CRISPR proteins

Stadelmann

Heid

Jendrusch

et al. 2021

Preprint

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Deep mutational scanning is a powerful method to explore the mutational fitness landscape of proteins. Its adaptation to anti-CRISPR proteins, which are natural CRISPR-Cas inhibitors and key players in the co-evolution of microbes and phages, would facilitate their in-depth characterization and optimization. Here, we developed a robust anti-CRISPR deep mutational scanning pipeline in Escherichia coli combining synthetic gene circuits based on CRISPR interference with flow cytometry-coupled sequencing and mathematical modeling. Using this pipeline, we created and characterized comprehensive single point mutation libraries for AcrIIA4 and AcrIIA5, two potent inhibitors of Streptococcus pyogenes Cas9. The resulting mutational fitness landscapes revealed that both Acrs possess a considerable mutational tolerance as well as an intrinsic redundancy with respect to Cas9 inhibitory features, suggesting evolutionary pressure towards high plasticity and robustness. Finally, to demonstrate that our pipeline can inform the optimization and fine-tuning of Acrs for genome editing applications, we cross-validated a subset of AcrIIA4 mutants via gene editing assays in mammalian cells and in vitro affinity measurements. Together, our work establishes deep mutational scanning as powerful method for anti-CRISPR protein characterization and optimization.

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“…Therefore, fine-tuning the expression of BEs and sgRNAs by using the optimal combination of promoters is a crucial factor in minimizing the toxic effects in the IRI-CCE platform. A recent study [44] indicated that the sufficiently perturbed dsDNA in bacteria-during replication, transcription, or other rearrangements-provides enough opportunity for Cas9 binding to a target site in the form of dynamically stretched single-strand DNA (ssDNA) or un-and underwound doublestrand DNA. Therefore, it is conceivable that the fewer nCas9-BE enzyme molecules, mainly evolved versions with a faster catalytic rate [18,45], produced by p35S-based promoters are enough to show relatively higher deamination activity.…”

Section: Discussionmentioning

confidence: 99%

In Vivo Rapid Investigation of CRISPR-Based Base Editing Components in Escherichia coli (IRI-CCE): A Platform for Evaluating Base Editing Tools and Their Components

Shelake

Pramanik

Kim

2022

IJMS

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Rapid assessment of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based genome editing (GE) tools and their components is a critical aspect for successful GE applications in different organisms. In many bacteria, double-strand breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient nonhomologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide (nt) editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs, cytidine base editor (CBE) and adenine base editor (ABE), capable of converting C to T and A to G, respectively, have been recently developed but yet to be tested for editing characteristics in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) comprising a combination of promoters and terminators enabling the expression of nCas9-based BE and sgRNA to nontoxic levels, eventually leading to successful base editing. We demonstrate the use of IRI-CCE to characterize different variants of CBEs (PmCDA1, evoCDA1, APOBEC3A) and ABEs (ABE8e, ABE9e) for bacteria, exhibiting that each independent BE has its specific editing pattern for a given target site depending on protospacer length. In summary, CRISPR-BE components expressed without lethal effects on cell survival in the IRI-CCE allow an analysis of various BE tools, including cloned biopart modules and sgRNAs.

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Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules

Cited by 6 publications

References 68 publications

Decoupling the bridge helix of Cas12a results in a reduced trimming activity, increased mismatch sensitivity and impaired conformational transitions

Decoupling the bridge helix of Cas12a results in a reduced trimming activity, increased mismatch sensitivity and impaired conformational transitions

A deep mutational scanning platform to characterize the fitness landscape of anti-CRISPR proteins

In Vivo Rapid Investigation of CRISPR-Based Base Editing Components in Escherichia coli (IRI-CCE): A Platform for Evaluating Base Editing Tools and Their Components

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