High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage

Ian M Slaymaker; Pablo Mesa; Max J Kellner; Soumya Kannan; Edward Brignole; Jeremy Koob; Patricia R Feliciano; Stefano Stella; Omar O Abudayyeh; Jonathan S Gootenberg; Jonathan Strecker; Guillermo Montoya; Feng Zhang

doi:10.1016/j.celrep.2019.02.094

High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage

Ian M Slaymaker, Pablo Mesa, Max J Kellner, Soumya Kannan, Edward Brignole, Jeremy Koob, Patricia R Feliciano, Stefano Stella, Omar O Abudayyeh, Jonathan S Gootenberg, Jonathan Strecker, Guillermo Montoya, Feng Zhang

Protein Structure and Function Program

18 Citations (Scopus)

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Abstract

Type VI CRISPR-Cas systems contain programmable single-effector RNA-guided RNases, including Cas13b, one of the four known family members. Cas13b, which has been used for both RNA editing and nucleic acid detection, is unique among type VI CRISPR effectors in its linear domain architecture and CRISPR RNA (crRNA) structure. Here, we report the crystal structure of Prevotella buccae Cas13b (PbuCas13b) bound to crRNA at 1.65 Å resolution. This structure, combined with biochemical experiments assaying the stability, kinetics, and function of Cas13b, provides a mechanistic model for Cas13b target RNA recognition and identifies features responsible for target and cleavage specificity. Based on these observations, we generated Cas13b variants with altered cleavage preferences, which may expand the utility of nuclease-based RNA detection assays and other applications of Cas13b in mammalian cells. Slaymaker et al. present a high-resolution structure of Cas13b in combination with biochemical studies showing that Cas13b is a highly dynamic, multi-turnover enzyme distinct from other Cas13 family members. Additionally, the authors demonstrate Cas13b can be rationally engineered to change RNA cleavage specificity and create a minimal variant suitable for delivery with viral vectors.

Original language	English
Journal	Cell Reports
Volume	26
Issue number	13
Pages (from-to)	3741-3751.e5
Number of pages	17
ISSN	2211-1247
DOIs	https://doi.org/10.1016/j.celrep.2019.02.094
Publication status	Published - 26 Mar 2019

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High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and CleavageFinal published version, 4.37 MBLicence: CC BY

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Slaymaker, I. M., Mesa, P., Kellner, M. J., Kannan, S., Brignole, E., Koob, J., Feliciano, P. R., Stella, S., Abudayyeh, O. O., Gootenberg, J. S., Strecker, J., Montoya, G., & Zhang, F. (2019). High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage. Cell Reports, 26(13), 3741-3751.e5. https://doi.org/10.1016/j.celrep.2019.02.094

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title = "High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage",

abstract = "Type VI CRISPR-Cas systems contain programmable single-effector RNA-guided RNases, including Cas13b, one of the four known family members. Cas13b, which has been used for both RNA editing and nucleic acid detection, is unique among type VI CRISPR effectors in its linear domain architecture and CRISPR RNA (crRNA) structure. Here, we report the crystal structure of Prevotella buccae Cas13b (PbuCas13b) bound to crRNA at 1.65 {\AA} resolution. This structure, combined with biochemical experiments assaying the stability, kinetics, and function of Cas13b, provides a mechanistic model for Cas13b target RNA recognition and identifies features responsible for target and cleavage specificity. Based on these observations, we generated Cas13b variants with altered cleavage preferences, which may expand the utility of nuclease-based RNA detection assays and other applications of Cas13b in mammalian cells. Slaymaker et al. present a high-resolution structure of Cas13b in combination with biochemical studies showing that Cas13b is a highly dynamic, multi-turnover enzyme distinct from other Cas13 family members. Additionally, the authors demonstrate Cas13b can be rationally engineered to change RNA cleavage specificity and create a minimal variant suitable for delivery with viral vectors.",

author = "Slaymaker, {Ian M} and Pablo Mesa and Kellner, {Max J} and Soumya Kannan and Edward Brignole and Jeremy Koob and Feliciano, {Patricia R} and Stefano Stella and Abudayyeh, {Omar O} and Gootenberg, {Jonathan S} and Jonathan Strecker and Guillermo Montoya and Feng Zhang",

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T1 - High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage

AU - Slaymaker, Ian M

AU - Mesa, Pablo

AU - Kellner, Max J

AU - Kannan, Soumya

AU - Brignole, Edward

AU - Koob, Jeremy

AU - Feliciano, Patricia R

AU - Stella, Stefano

AU - Abudayyeh, Omar O

AU - Gootenberg, Jonathan S

AU - Strecker, Jonathan

AU - Montoya, Guillermo

AU - Zhang, Feng

PY - 2019/3/26

Y1 - 2019/3/26

N2 - Type VI CRISPR-Cas systems contain programmable single-effector RNA-guided RNases, including Cas13b, one of the four known family members. Cas13b, which has been used for both RNA editing and nucleic acid detection, is unique among type VI CRISPR effectors in its linear domain architecture and CRISPR RNA (crRNA) structure. Here, we report the crystal structure of Prevotella buccae Cas13b (PbuCas13b) bound to crRNA at 1.65 Å resolution. This structure, combined with biochemical experiments assaying the stability, kinetics, and function of Cas13b, provides a mechanistic model for Cas13b target RNA recognition and identifies features responsible for target and cleavage specificity. Based on these observations, we generated Cas13b variants with altered cleavage preferences, which may expand the utility of nuclease-based RNA detection assays and other applications of Cas13b in mammalian cells. Slaymaker et al. present a high-resolution structure of Cas13b in combination with biochemical studies showing that Cas13b is a highly dynamic, multi-turnover enzyme distinct from other Cas13 family members. Additionally, the authors demonstrate Cas13b can be rationally engineered to change RNA cleavage specificity and create a minimal variant suitable for delivery with viral vectors.

AB - Type VI CRISPR-Cas systems contain programmable single-effector RNA-guided RNases, including Cas13b, one of the four known family members. Cas13b, which has been used for both RNA editing and nucleic acid detection, is unique among type VI CRISPR effectors in its linear domain architecture and CRISPR RNA (crRNA) structure. Here, we report the crystal structure of Prevotella buccae Cas13b (PbuCas13b) bound to crRNA at 1.65 Å resolution. This structure, combined with biochemical experiments assaying the stability, kinetics, and function of Cas13b, provides a mechanistic model for Cas13b target RNA recognition and identifies features responsible for target and cleavage specificity. Based on these observations, we generated Cas13b variants with altered cleavage preferences, which may expand the utility of nuclease-based RNA detection assays and other applications of Cas13b in mammalian cells. Slaymaker et al. present a high-resolution structure of Cas13b in combination with biochemical studies showing that Cas13b is a highly dynamic, multi-turnover enzyme distinct from other Cas13 family members. Additionally, the authors demonstrate Cas13b can be rationally engineered to change RNA cleavage specificity and create a minimal variant suitable for delivery with viral vectors.

U2 - 10.1016/j.celrep.2019.02.094

DO - 10.1016/j.celrep.2019.02.094

M3 - Journal article

C2 - 30917325

SN - 2211-1247

VL - 26

SP - 3741-3751.e5

JO - Cell Reports

JF - Cell Reports

IS - 13

ER -

High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage

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