CRISPR-Cas12a Nucleases Function with Structurally Engineered CrRNAs: SynThetic TrAcrRNA

Overview

Journal Sci Rep

Specialty Science

Date 2022 Jul 16

PMID 35842430

Authors

D J Jedrzejczyk

L D Poulsen

M Mohr

N D Damas

S Schoffelen

A Barghetti

R Baumgartner

B T Weinert

T Warnecke

R T Gill

Affiliations

Soon will be listed here.

Abstract

CRISPR-Cas12a systems are becoming an attractive genome editing tool for cell engineering due to their broader editing capabilities compared to CRISPR-Cas9 counterparts. As opposed to Cas9, the Cas12a endonucleases are characterized by a lack of trans-activating crRNA (tracrRNA), which reduces the complexity of the editing system and simultaneously makes CRISPR RNA (crRNA) engineering a promising approach toward further improving and modulating editing activity of the CRISPR-Cas12a systems. Here, we design and validate sixteen types of structurally engineered Cas12a crRNAs targeting various immunologically relevant loci in-vitro and in-cellulo. We show that all our structural modifications in the loop region, ranging from engineered breaks (STAR-crRNAs) to large gaps (Gap-crRNAs), as well as nucleotide substitutions, enable gene-cutting in the presence of various Cas12a nucleases. Moreover, we observe similar insertion rates of short HDR templates using the engineered crRNAs compared to the wild-type crRNAs, further demonstrating that the introduced modifications in the loop region led to comparable genome editing efficiencies. In conclusion, we show that Cas12a nucleases can broadly utilize structurally engineered crRNAs with breaks or gaps in the otherwise highly-conserved loop region, which could further facilitate a wide range of genome editing applications.

Citing Articles

Synthetic mismatches enable specific CRISPR-Cas12a-based detection of genome-wide SNVs tracked by ARTEMIS.

Kohabir K, Linthorst J, Nooi L, Brouwer R, Wolthuis R, Sistermans E Cell Rep Methods. 2024; 4(12):100912.

PMID: 39644903 PMC: 11704620. DOI: 10.1016/j.crmeth.2024.100912.

Interpreting CRISPR-Cas12a enzyme kinetics through free energy change of nucleic acids.

Zhang J, Guan X, Moon J, Zhang S, Jia Z, Yang R Nucleic Acids Res. 2024; 52(22):14077-14092.

PMID: 39588774 PMC: 11662684. DOI: 10.1093/nar/gkae1124.

Iterative crRNA design and a PAM-free strategy enabled an ultra-specific RPA-CRISPR/Cas12a detection platform.

Mao X, Xu J, Jiang J, Li Q, Yao P, Jiang J Commun Biol. 2024; 7(1):1454.

PMID: 39506042 PMC: 11541961. DOI: 10.1038/s42003-024-07173-7.

Insight into the natural regulatory mechanisms and clinical applications of the CRISPR-Cas system.

Cheng H, Deng H, Ma D, Gao M, Zhou Z, Li H Heliyon. 2024; 10(20):e39538.

PMID: 39502233 PMC: 11535992. DOI: 10.1016/j.heliyon.2024.e39538.

Kinetic dissection of pre-crRNA binding and processing by CRISPR-Cas12a.

Sinan S, Appleby N, Chou C, Finkelstein I, Russell R RNA. 2024; 30(10):1345-1355.

PMID: 39009379 PMC: 11404446. DOI: 10.1261/rna.080088.124.

References

Andrews S, Gilley J, Coleman M . Difference Tracker: ImageJ plugins for fully automated analysis of multiple axonal transport parameters. J Neurosci Methods. 2010; 193(2):281-7. DOI: 10.1016/j.jneumeth.2010.09.007. View

Kleinstiver B, Sousa A, Walton R, Tak Y, Hsu J, Clement K . Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat Biotechnol. 2019; 37(3):276-282. PMC: 6401248. DOI: 10.1038/s41587-018-0011-0. View

Makarova K, Wolf Y, Koonin E . Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?. CRISPR J. 2019; 1(5):325-336. PMC: 6636873. DOI: 10.1089/crispr.2018.0033. View

Sander J, Joung J . CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol. 2014; 32(4):347-55. PMC: 4022601. DOI: 10.1038/nbt.2842. View

Mohanraju P, van der Oost J, Jinek M, Swarts D . Heterologous Expression and Purification of CRISPR-Cas12a/Cpf1. Bio Protoc. 2021; 8(9):e2842. PMC: 8275269. DOI: 10.21769/BioProtoc.2842. View

Shmakov S, Smargon A, Scott D, Cox D, Pyzocha N, Yan W . Diversity and evolution of class 2 CRISPR-Cas systems. Nat Rev Microbiol. 2017; 15(3):169-182. PMC: 5851899. DOI: 10.1038/nrmicro.2016.184. View

Couvin D, Bernheim A, Toffano-Nioche C, Touchon M, Michalik J, Neron B . CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins. Nucleic Acids Res. 2018; 46(W1):W246-W251. PMC: 6030898. DOI: 10.1093/nar/gky425. View

Wu H, Liu Q, Shi H, Xie J, Zhang Q, Ouyang Z . Engineering CRISPR/Cpf1 with tRNA promotes genome editing capability in mammalian systems. Cell Mol Life Sci. 2018; 75(19):3593-3607. PMC: 11105780. DOI: 10.1007/s00018-018-2810-3. View

Nishimasu H, Yamano T, Gao L, Zhang F, Ishitani R, Nureki O . Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1. Mol Cell. 2017; 67(1):139-147.e2. PMC: 5957533. DOI: 10.1016/j.molcel.2017.04.019. View

10.

Clement K, Rees H, Canver M, Gehrke J, Farouni R, Hsu J . CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat Biotechnol. 2019; 37(3):224-226. PMC: 6533916. DOI: 10.1038/s41587-019-0032-3. View

11.

Fu Y, Sander J, Reyon D, Cascio V, Joung J . Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol. 2014; 32(3):279-284. PMC: 3988262. DOI: 10.1038/nbt.2808. View

12.

Zhang Q, Ye Y . Not all predicted CRISPR-Cas systems are equal: isolated cas genes and classes of CRISPR like elements. BMC Bioinformatics. 2017; 18(1):92. PMC: 5294841. DOI: 10.1186/s12859-017-1512-4. View

13.

Makarova K, Haft D, Barrangou R, Brouns S, Charpentier E, Horvath P . Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol. 2011; 9(6):467-77. PMC: 3380444. DOI: 10.1038/nrmicro2577. View

14.

Kocak D, Josephs E, Bhandarkar V, Adkar S, Kwon J, Gersbach C . Increasing the specificity of CRISPR systems with engineered RNA secondary structures. Nat Biotechnol. 2019; 37(6):657-666. PMC: 6626619. DOI: 10.1038/s41587-019-0095-1. View

15.

Gao L, Cox D, Yan W, Manteiga J, Schneider M, Yamano T . Engineered Cpf1 variants with altered PAM specificities. Nat Biotechnol. 2017; 35(8):789-792. PMC: 5548640. DOI: 10.1038/nbt.3900. View

16.

Strecker J, Jones S, Koopal B, Schmid-Burgk J, Zetsche B, Gao L . Engineering of CRISPR-Cas12b for human genome editing. Nat Commun. 2019; 10(1):212. PMC: 6342934. DOI: 10.1038/s41467-018-08224-4. View

17.

Yan W, Hunnewell P, Alfonse L, Carte J, Keston-Smith E, Sothiselvam S . Functionally diverse type V CRISPR-Cas systems. Science. 2018; 363(6422):88-91. PMC: 11258546. DOI: 10.1126/science.aav7271. View

18.

Shebanova R, Nikitchina N, Shebanov N, Mekler V, Kuznedelov K, Ulashchik E . Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA. Nucleic Acids Res. 2021; 50(2):1162-1173. PMC: 8789034. DOI: 10.1093/nar/gkab1227. View

19.

Zetsche B, Gootenberg J, Abudayyeh O, Slaymaker I, Makarova K, Essletzbichler P . Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015; 163(3):759-71. PMC: 4638220. DOI: 10.1016/j.cell.2015.09.038. View

20.

Liu R, Liang L, Freed E, Chang H, Oh E, Liu Z . Synthetic chimeric nucleases function for efficient genome editing. Nat Commun. 2019; 10(1):5524. PMC: 6892893. DOI: 10.1038/s41467-019-13500-y. View