Programmed DNA Destruction by Miniature CRISPR-Cas14 Enzymes

Overview

Journal Science

Specialty Science

Date 2018 Oct 20

PMID 30337455

Citations 398

Authors

Lucas B Harrington

David Burstein

Janice S Chen

David Paez-Espino

Enbo Ma

Isaac P Witte

Joshua C Cofsky

Nikos C Kyrpides

Jillian F Banfield

Jennifer A Doudna

Affiliations

Soon will be listed here.

Abstract

CRISPR-Cas systems provide microbes with adaptive immunity to infectious nucleic acids and are widely employed as genome editing tools. These tools use RNA-guided Cas proteins whose large size (950 to 1400 amino acids) has been considered essential to their specific DNA- or RNA-targeting activities. Here we present a set of CRISPR-Cas systems from uncultivated archaea that contain Cas14, a family of exceptionally compact RNA-guided nucleases (400 to 700 amino acids). Despite their small size, Cas14 proteins are capable of targeted single-stranded DNA (ssDNA) cleavage without restrictive sequence requirements. Moreover, target recognition by Cas14 triggers nonspecific cutting of ssDNA molecules, an activity that enables high-fidelity single-nucleotide polymorphism genotyping (Cas14-DETECTR). Metagenomic data show that multiple CRISPR-Cas14 systems evolved independently and suggest a potential evolutionary origin of single-effector CRISPR-based adaptive immunity.

Citing Articles

Nanotechnology-leveraged CRISPR/Cas systems: icebreaking in trace cancer-related nucleic acids biosensing.

Peng W, Shi M, Hu B, Jia J, Li X, Wang N Mol Cancer. 2025; 24(1):78.

PMID: 40087758 DOI: 10.1186/s12943-024-02222-5.

Efforts to Downsize Base Editors for Clinical Applications.

Song B Int J Mol Sci. 2025; 26(5).

PMID: 40076976 PMC: 11900391. DOI: 10.3390/ijms26052357.

A review of gut failure as a cause and consequence of critical illness.

Soranno D, Coopersmith C, Brinkworth J, Factora F, Muntean J, Mythen M Crit Care. 2025; 29(1):91.

PMID: 40011975 PMC: 11866815. DOI: 10.1186/s13054-025-05309-7.

From Origin to the Present: Establishment, Mechanism, Evolutions and Biomedical Applications of the CRISPR/Cas-Based Macromolecular System in Brief.

Yuan Z Molecules. 2025; 30(4).

PMID: 40005257 PMC: 11858448. DOI: 10.3390/molecules30040947.

Exploiting the Specificity of CRISPR/Cas System for Nucleic Acids Amplification-Free Disease Diagnostics in the Point-of-Care.

Yee B, Ali N, Mohd-Naim N, Ahmed M Chem Bio Eng. 2025; 1(4):330-339.

PMID: 39974464 PMC: 11835143. DOI: 10.1021/cbe.3c00112.

References

Li S, Cheng Q, Liu J, Nie X, Zhao G, Wang J . CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA. Cell Res. 2018; 28(4):491-493. PMC: 5939048. DOI: 10.1038/s41422-018-0022-x. View

Letunic I, Bork P . Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res. 2016; 44(W1):W242-5. PMC: 4987883. DOI: 10.1093/nar/gkw290. View

Abudayyeh O, Gootenberg J, Konermann S, Joung J, Slaymaker I, Cox D . C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science. 2016; 353(6299):aaf5573. PMC: 5127784. DOI: 10.1126/science.aaf5573. View

Biswas A, Staals R, Morales S, Fineran P, Brown C . CRISPRDetect: A flexible algorithm to define CRISPR arrays. BMC Genomics. 2016; 17:356. PMC: 4869251. DOI: 10.1186/s12864-016-2627-0. View

Liu L, Li X, Ma J, Li Z, You L, Wang J . The Molecular Architecture for RNA-Guided RNA Cleavage by Cas13a. Cell. 2017; 170(4):714-726.e10. DOI: 10.1016/j.cell.2017.06.050. View

Zhang Y, Rajan R, Seifert H, Mondragon A, Sontheimer E . DNase H Activity of Neisseria meningitidis Cas9. Mol Cell. 2015; 60(2):242-55. PMC: 4609032. DOI: 10.1016/j.molcel.2015.09.020. View

Anantharaman K, Brown C, Hug L, Sharon I, Castelle C, Probst A . Thousands of microbial genomes shed light on interconnected biogeochemical processes in an aquifer system. Nat Commun. 2016; 7:13219. PMC: 5079060. DOI: 10.1038/ncomms13219. View

Burstein D, Harrington L, Strutt S, Probst A, Anantharaman K, Thomas B . New CRISPR-Cas systems from uncultivated microbes. Nature. 2016; 542(7640):237-241. PMC: 5300952. DOI: 10.1038/nature21059. View

Harrington L, Paez-Espino D, Staahl B, Chen J, Ma E, Kyrpides N . A thermostable Cas9 with increased lifetime in human plasma. Nat Commun. 2017; 8(1):1424. PMC: 5681539. DOI: 10.1038/s41467-017-01408-4. View

10.

Ogata H, Goto S, Sato K, Fujibuchi W, Bono H, Kanehisa M . KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 1998; 27(1):29-34. PMC: 148090. DOI: 10.1093/nar/27.1.29. View

11.

Markowitz V, Chen I, Palaniappan K, Chu K, Szeto E, Grechkin Y . IMG: the Integrated Microbial Genomes database and comparative analysis system. Nucleic Acids Res. 2011; 40(Database issue):D115-22. PMC: 3245086. DOI: 10.1093/nar/gkr1044. View

12.

Deltcheva E, Chylinski K, Sharma C, Gonzales K, Chao Y, Pirzada Z . CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011; 471(7340):602-7. PMC: 3070239. DOI: 10.1038/nature09886. View

13.

Shmakov S, Abudayyeh O, Makarova K, Wolf Y, Gootenberg J, Semenova E . Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Mol Cell. 2015; 60(3):385-97. PMC: 4660269. DOI: 10.1016/j.molcel.2015.10.008. View

14.

Mojica F, Diez-Villasenor C, Garcia-Martinez J, Almendros C . Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology (Reading). 2009; 155(Pt 3):733-740. DOI: 10.1099/mic.0.023960-0. View

15.

Chen J, Ma E, Harrington L, Da Costa M, Tian X, Palefsky J . CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. 2018; 360(6387):436-439. PMC: 6628903. DOI: 10.1126/science.aar6245. View

16.

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

17.

Koonin E, Makarova K, Zhang F . Diversity, classification and evolution of CRISPR-Cas systems. Curr Opin Microbiol. 2017; 37:67-78. PMC: 5776717. DOI: 10.1016/j.mib.2017.05.008. View

18.

Yoshida M, Mochizuki T, Urayama S, Yoshida-Takashima Y, Nishi S, Hirai M . Quantitative Viral Community DNA Analysis Reveals the Dominance of Single-Stranded DNA Viruses in Offshore Upper Bathyal Sediment from Tohoku, Japan. Front Microbiol. 2018; 9:75. PMC: 5807898. DOI: 10.3389/fmicb.2018.00075. View

19.

Stamatakis A . RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014; 30(9):1312-3. PMC: 3998144. DOI: 10.1093/bioinformatics/btu033. View

20.

Castelle C, Wrighton K, Thomas B, Hug L, Brown C, Wilkins M . Genomic expansion of domain archaea highlights roles for organisms from new phyla in anaerobic carbon cycling. Curr Biol. 2015; 25(6):690-701. DOI: 10.1016/j.cub.2015.01.014. View