Closely related type II-C Cas9 orthologs recognize diverse PAMs

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    Evaluation Summary:

    This work is relevant to all who are interested in genome editing. The versatile Cas9 nuclease has enabled creative genome editing applications, yet the targetable sequence space is limited by the PAM specificity of the Cas9 RNP. This manuscript expands the Cas9 toolbox by defining the PAM specificity and genome editing activity of a large group of smaller-sized type II-C Cas9s. The results also contribute to our understanding of the diversity of Cas enzymes and show that there is a significant potential in mining for non-trivial genome editing tools amongst highly similar Cas orthologs.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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Abstract

The RNA-guided CRISPR/Cas9 system is a powerful tool for genome editing, but its targeting scope is limited by the protospacer-adjacent motif (PAM). To expand the target scope, it is crucial to develop a CRISPR toolbox capable of recognizing multiple PAMs. Here, using a GFP-activation assay, we tested the activities of 29 type II-C orthologs closely related to Nme1Cas9, 25 of which are active in human cells. These orthologs recognize diverse PAMs with variable length and nucleotide preference, including purine-rich, pyrimidine-rich, and mixed purine and pyrimidine PAMs. We characterized in depth the activity and specificity of Nsp2Cas9. We also generated a chimeric Cas9 nuclease that recognizes a simple N 4 C PAM, representing the most relaxed PAM preference for compact Cas9s to date. These Cas9 nucleases significantly enhance our ability to perform allele-specific genome editing.

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  1. Author Response

    Reviewer #1 (Public Review):

    The tools and approaches in this manuscript are of broad interest, not only to protein engineers but also to the many researchers using genome-editing reagents. However, putting the work in the context of previous research, both through changing the writing and additional experiments, will be critical for taking advantage of that widespread applicability.

    Strengths:

    Overall, the data support the conclusions of the manuscript.

    The most exciting product of this work is an engineered nuclease, Nsp2-SmuCas9, that has high activity and specificity in human cells and a relaxed PAM preference for a single C base. This chimeric enzyme can efficiently induce indels at endogenous sites. While other works have presented nucleases with minimal PAM preferences, Nsp2-SmuCas9 is a useful …

  2. Evaluation Summary:

    This work is relevant to all who are interested in genome editing. The versatile Cas9 nuclease has enabled creative genome editing applications, yet the targetable sequence space is limited by the PAM specificity of the Cas9 RNP. This manuscript expands the Cas9 toolbox by defining the PAM specificity and genome editing activity of a large group of smaller-sized type II-C Cas9s. The results also contribute to our understanding of the diversity of Cas enzymes and show that there is a significant potential in mining for non-trivial genome editing tools amongst highly similar Cas orthologs.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the …

  3. Reviewer #1 (Public Review):

    The tools and approaches in this manuscript are of broad interest, not only to protein engineers but also to the many researchers using genome-editing reagents. However, putting the work in the context of previous research, both through changing the writing and additional experiments, will be critical for taking advantage of that widespread applicability.

    Strengths:

    Overall, the data support the conclusions of the manuscript.

    The most exciting product of this work is an engineered nuclease, Nsp2-SmuCas9, that has high activity and specificity in human cells and a relaxed PAM preference for a single C base. This chimeric enzyme can efficiently induce indels at endogenous sites. While other works have presented nucleases with minimal PAM preferences, Nsp2-SmuCas9 is a useful alternative and may be preferred. …

  4. Reviewer #2 (Public Review):

    The versatile Cas9 has enabled creative genome editing applications. The targetable sequence space is limited by the PAM specificity of the Cas9 RNP. In this manuscript, the authors made a comprehensive attempt to explore the genome editing potential of the under-explored type II-C Cas9s. A positive selection assay was set up to define the PAM specificity of twenty nine different II-C Cas9 homologs. Twenty five of them were active in genome editing in human cells. Their distinct PAM specificities can be rationalized to some extent based on the identity of the key residues in the PAM-interaction domain. The authors then focused on Nsp2Cas9 and a few others. Nsp2Cas9 can be engineered to recognize a single C PAM by replacing the PI domain with that of the SmuCas9. Impressively, the resulting chimera retained …

  5. Reviewer #3 (Public Review):

    Searches for new types of CRISPR-Cas nucleases bring back enzymes with unique properties. Characterization of closely related Cas homologues can also be useful and result in discoveries of enzymes with properties that complement those of existing editors. The large-scale characterization of Cas9 orthologs by the Siksnys group, research by the Sontheimer, Zhang, and Doudna groups, as well as work from other laboratories, resulted in characterization of Cas9 enzymes which recognize distinct PAMs and can be used to edit a wide range of genomic targets. The authors of this paper contributed to the effort of expanding the Cas9 toolbox by characterizing BlatCas9 and SauriCas9, two enzymes that may be promising for genome engineering.

    The main difficulty with most Cas9 orthologs is their low activity in human …