CRISPR-Cas adaptive immune systems of bacteria and archaea have catapulted into the scientific spotlight as genome editing tools. To aid researchers in the field, we have developed an automated pipeline, named CRISPRdisco (CRISPR discovery), to identify CRISPR repeats and cas genes in genome assemblies, determine type and subtype, and describe system completeness. All six major types and 23 currently recognized subtypes and novel putative V-U types are detected. Here, we use the pipeline to identify and classify putative CRISPR-Cas systems in 2,777 complete genomes from the NCBI RefSeq database. This allows comparison to previous publications and investigation of the occurrence and size of CRISPR-Cas systems. Software available at http://github.com/crisprlab/CRISPRdisco provides reproducible, standardized, accessible, transparent, and high-throughput analysis methods available to all researchers in and beyond the CRISPR-Cas research community. This tool opens new avenues to enable classification within a complex nomenclature and provides analytical methods in a field that has evolved rapidly.
Get full access to this article
View all access options for this article.
References
1.
JacksonSA, McKenzieRE, FagerlundRD, et al.CRISPR-Cas: adapting to change. Science, 2017; 356. DOI: 10.1126/science.aal5055.
2.
KnightS, TjianR, DoudnaJ.Genomes in focus: development and applications of Crispr-Cas9 imaging technologies. Angew Chem Int Ed Engl, 2017 Oct 27 [Epub ahead of print]; DOI: 10.1002/anie.201709201.
3.
BarrangouR, HorvathP. A decade of discovery: CRISPR functions and applications. Nat Microbiol, 2017; 2:1709–2. DOI: 10.1038/nmicrobiol.2017.92
4.
KooninEV, MakarovaKS, ZhangF. Diversity, classification and evolution of CRISPR-Cas systems. Curr Opin Microbiol, 2017; 37:67–78. DOI: 10.1016/j.mib.2017.05.008.
5.
ShmakovS, SmargonA, ScottD, et al.Diversity and evolution of class 2 CRISPR-Cas systems. Nat Rev Microbiol, 2017; 15:169–182. DOI: 10.1038/nrmicro.2016.184.
ShmakovS, AbudayyehOO, MakarovaKS, et al.Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Mol Cell, 2015; 60:385–397. DOI: 10.1016/j.molcel.2015.10.008.
9.
BursteinD, HarringtonLB, StruttSC, et al.New CRISPR-Cas systems from uncultivated microbes. Nature, 2017; 542:237–241. DOI: 10.1038/nature21059.
10.
MakarovaKS, WolfYI, AlkhnbashiOS, et al.An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol, 2015; 13:722–736. DOI: 10.1038/nrmicro3569.
11.
BlandC, RamseyTL, SabreeF, et al.CRISPR recognition tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats. BMC Bioinformatics, 2007; 8:20–9. DOI: 10.1186/1471-2105-8-209.
12.
GrissaI, VergnaudG, PourcelC. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res, 2007; 35:W52–57. DOI: 10.1093/nar/gkm360.
13.
AlkhnbashiOS, CostaF, ShahSA, et al.CRISPRstrand: predicting repeat orientations to determine the crRNA-encoding strand at CRISPR loci. Bioinformatics, 2014; 30:i489–496. DOI: 10.1093/bioinformatics/btu459.
14.
AlkhnbashiOS, ShahSA, GarrettRA, et al.Characterizing leader sequences of CRISPR loci. Bioinformatics, 2016; 32:i576–i585. DOI: 10.1093/bioinformatics/btw454.
15.
WeiY, ChesneMT, TernsRM, et al.Sequences spanning the leader-repeat junction mediate CRISPR adaptation to phage in Streptococcus thermophilus. Nucleic Acids Res, 2015; 43:1749–1758. DOI: 10.1093/nar/gku1407.
16.
TomsA, BarrangouR. On the global CRISPR array behavior in class I systems. Biol Direct, 2017; 12:2–0. DOI: 10.1186/s13062-017-0193-2.
17.
BiswasA, StaalsRH, MoralesSE, et al.CRISPRDetect: a flexible algorithm to define CRISPR arrays. BMC Genomics, 2016; 17:35–6. DOI: 10.1186/s12864-016-2627-0.
18.
FonfaraI, Le RhunA, ChylinskiK, et al.Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Res, 2014; 42:2577–2590. DOI: 10.1093/nar/gkt1074.
19.
ChylinskiK, Le RhunA, CharpentierE. The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems. RNA Biol, 2013; 10:726–737. DOI: 10.4161/rna.24321.
20.
CarteJ, ChristopherRT, SmithJT, et al.The three major types of CRISPR-Cas systems function independently in CRISPR RNA biogenesis in Streptococcus thermophilus. Mol Microbiol, 2014; 93:98–112. DOI: 10.1111/mmi.12644.
21.
PrinsP, de LigtJ, TarasovA, et al.Toward effective software solutions for big biology. Nat Biotechnol, 2015; 33:686–687. DOI: 10.1038/nbt.3240.
22.
PengRD.Reproducible research in computational science. Science, 2011; 334:1226–1227. DOI: 10.1126/science.1213847.
23.
GruningBA, RascheE, Rebolledo-JaramilloB, et al.Jupyter and Galaxy: easing entry barriers into complex data analyses for biomedical researchers. PLoS Comput Biol, 2017; 13:e100542–5. DOI: 10.1371/journal.pcbi.1005425.
24.
ShenH.Interactive notebooks: sharing the code. Nature, 2014; 515:151–152. DOI: 10.1038/515151a.
BoettigerC.An introduction to Docker for reproducible research. SIGOPS Oper Syst Rev, 2015; 49:71–79. DOI: 10.1145/2723872.2723882.
27.
HHMI. hmmscan: search sequence(s) against a profile database. http://hmmer.org/ (last accessed December2017).
28.
BeloglazovaN, PetitP, FlickR, et al.Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference. EMBO J, 2011; 30:4616–4627. DOI: 10.1038/emboj.2011.377.
29.
MulepatiS, BaileyS. Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3). J Biol Chem, 2011; 286:31896–31903. DOI: 10.1074/jbc.M111.270017.
30.
CadyKC, O'TooleGA. Non-identity-mediated CRISPR-bacteriophage interaction mediated via the Csy and Cas3 proteins. J Bacteriol, 2011; 193:3433–3445. DOI: 10.1128/JB.01411-10.
31.
SinkunasT, GasiunasG, FremauxC, et al.Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J, 2011; 30:1335–1342. DOI: 10.1038/emboj.2011.41.
32.
DeltchevaE, ChylinskiK, SharmaCM, et al.CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature, 2011; 471:602–607. DOI: 10.1038/nature09886.
33.
GasiunasG, BarrangouR, HorvathP, et al.Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A, 2012; 109:E2579–2586. DOI: 10.1073/pnas.1208507109.
34.
SapranauskasR, GasiunasG, FremauxC, et al.The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res, 2011; 39:9275–9282. DOI: 10.1093/nar/gkr606.
35.
WalkerFC, Chou-ZhengL, DunkleJA, et al.Molecular determinants for CRISPR RNA maturation in the Cas10–Csm complex and roles for non-Cas nucleases. Nucleic Acids Res, 2017; 45:2112–2123. DOI: 10.1093/nar/gkw891.
36.
HaleCR, CocozakiA, LiH, et al.Target RNA capture and cleavage by the Cmr type III-B CRISPR-Cas effector complex. Genes Dev, 2014; 28:2432–2443. DOI: 10.1101/gad.250712.114.
37.
IchikawaHT, CooperJC, LoL, et al.Programmable type III-A CRISPR-Cas DNA targeting modules. PLoS One, 2017; 12:e017622–1. DOI: 10.1371/journal.pone.0176221.
38.
ZetscheB, GootenbergJS, AbudayyehOO, et al.Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 2015; 163:759–771. DOI: 10.1016/j.cell.2015.09.038.
39.
WuD, GuanX, ZhuY, et al.Structural basis of stringent PAM recognition by CRISPR-C2c1 in complex with sgRNA. Cell Res, 2017; 27:705–708. DOI: 10.1038/cr.2017.46.
40.
LiuL, ChenP, WangM, et al.C2c1–sgRNA complex structure reveals RNA-guided DNA cleavage mechanism. Mol Cell, 2017; 65:310–322. DOI: 10.1016/j.molcel.2016.11.040.
41.
YangH, GaoP, RajashankarKR, et al.PAM-dependent target DNA recognition and cleavage by C2c1 CRISPR-Cas endonuclease. Cell, 2016; 167:1814–1828.e12. DOI: 10.1016/j.cell.2016.11.053.
42.
CoxDBT, GootenbergJS, AbudayyehOO, et al.RNA editing with CRISPR-Cas13. Science, 2017; 358:1019–1027. DOI: 10.1126/science.aaq0180.
43.
AbudayyehOO, GootenbergJS, EssletzbichlerP, et al.RNA targeting with CRISPR-Cas13. Nature, 2017; 550:280–284. DOI: 10.1038/nature24049.
44.
AbudayyehOO, GootenbergJS, KonermannS, et al.C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science, 2016; 353:aaf557–3. DOI: 10.1126/science.aaf5573.
45.
GrissaI, VergnaudG, PourcelC. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics, 2007; 8:17–2. DOI: 10.1186/1471-2105-8-172.
46.
BursteinD, SunCL, BrownCT, et al.Major bacterial lineages are essentially devoid of CRISPR-Cas viral defence systems. Nat Commun, 2016; 7:1061–3. DOI: 10.1038/ncomms10613.
47.
SunCL, ThomasBC, BarrangouR, et al.Metagenomic reconstructions of bacterial CRISPR loci constrain population histories. ISME J, 2016; 10:858–870. DOI: 10.1038/ismej.2015.162.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
