A TXTL-Based Assay to Rapidly Identify PAMs for CRISPR-Cas Systems with Multi-Protein Effector Complexes.
Binding assay
CRISPR-Cas systems
Multi-protein complex
PAM
Positive screen
TXTL
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2022
2022
Historique:
entrez:
5
1
2022
pubmed:
6
1
2022
medline:
4
3
2022
Statut:
ppublish
Résumé
Type I CRISPR-Cas systems represent the most common and diverse type of these prokaryotic defense systems and are being harnessed for a growing set of applications. As these systems rely on multi-protein effector complexes, their characterization remains challenging. Here, we report a rapid and straightforward method to characterize these systems in a cell-free transcription-translation (TXTL) system. A ribonucleoprotein complex is produced and binds to its target next to a recognized PAM, thereby preventing the targeted sequence from being cleaved by a restriction enzyme. Selection for uncleaved targeted plasmids leads to an enrichment of recognized sequences within a PAM library. This assay will aid the exploration of CRISPR-Cas diversity and evolution and help contribute new systems for CRISPR technologies and applications.
Identifiants
pubmed: 34985758
doi: 10.1007/978-1-0716-1998-8_24
doi:
Types de publication
Journal Article
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
391-411Informations de copyright
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
Références
Barrangou R, Fremaux C, Deveau H et al (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712
pubmed: 17379808
Sorek R, Lawrence CM, Wiedenheft B (2013) CRISPR-mediated adaptive immune systems in bacteria and archaea. Annu Rev Biochem 82:237–266
pubmed: 23495939
Heler R, Marraffini LA, Bikard D (2014) Adapting to new threats: the generation of memory by CRISPR-Cas immune systems. Mol Microbiol 93:1–9
pubmed: 24806524
pmcid: 4104294
Yosef I, Shitrit D, Goren MG et al (2013) DNA motifs determining the efficiency of adaptation into the Escherichia coli CRISPR array. Proc Natl Acad Sci U S A 110:14396–14401
pubmed: 23940313
pmcid: 3761565
Wang J, Li J, Zhao H et al (2015) Structural and mechanistic basis of PAM-dependent spacer acquisition in CRISPR-Cas systems. Cell 163:840–853
pubmed: 26478180
Charpentier E, Richter H, van der Oost J, White MF (2015) Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity. FEMS Microbiol Rev 39:428–441
pubmed: 25994611
pmcid: 5965381
Leenay RT, Beisel CL (2017) Deciphering, communicating, and engineering the CRISPR PAM. J Mol Biol 429:177–191
pubmed: 27916599
Marraffini LA, Sontheimer EJ (2010) CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet 11:181–190
pubmed: 20125085
pmcid: 2928866
Makarova KS, Wolf YI, Iranzo J et al (2020) Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol 18:67–83
pubmed: 31857715
Brouns SJJ, Jore MM, Lundgren M et al (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321:960–964
pubmed: 18703739
pmcid: 5898235
Westra ER, van Erp PBG, Künne T et al (2012) CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol Cell 46:595–605
pubmed: 22521689
pmcid: 3372689
Csörgő B, León LM, Chau-Ly IJ et al (2020) A compact Cascade-Cas3 system for targeted genome engineering. Nat Methods 17:1183–1190
pubmed: 33077967
pmcid: 7611934
Chen Y, Liu J, Zhi S et al (2020) Repurposing type I–F CRISPR–Cas system as a transcriptional activation tool in human cells. Nat Commun 11:1–14
Dolan AE, Hou Z, Xiao Y et al (2019) Introducing a spectrum of long-range genomic deletions in human embryonic stem cells using type I CRISPR-Cas. Mol Cell 74:936–950.e5
pubmed: 30975459
pmcid: 6555677
Xu Z, Li M, Li Y et al (2019) Native CRISPR-Cas-mediated genome editing enables dissecting and sensitizing clinical multidrug-resistant P. aeruginosa. Cell Rep 29:1707–1717.e3
pubmed: 31693906
Luo ML, Mullis AS, Leenay RT, Beisel CL (2015) Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression. Nucleic Acids Res 43:674–681
pubmed: 25326321
Hidalgo-Cantabrana C, Barrangou R (2020) Characterization and applications of Type I CRISPR-Cas systems. Biochem Soc Trans 48:15–23
pubmed: 31922192
Morisaka H, Yoshimi K, Okuzaki Y et al (2019) CRISPR-Cas3 induces broad and unidirectional genome editing in human cells. Nat Commun 10:5302
pubmed: 31811138
pmcid: 6897959
Cheng F, Gong L, Zhao D et al (2017) Harnessing the native type I-B CRISPR-Cas for genome editing in a polyploid archaeon. J Genet Genomics 44:541–548
pubmed: 29169919
Li Y, Pan S, Zhang Y et al (2016) Harnessing type I and type III CRISPR-Cas systems for genome editing. Nucleic Acids Res 44:e34
pubmed: 26467477
Cameron P, Coons MM, Klompe SE et al (2019) Harnessing type I CRISPR-Cas systems for genome engineering in human cells. Nat Biotechnol 37:1471–1477
pubmed: 31740839
Pickar-Oliver A, Black JB, Lewis MM et al (2019) Targeted transcriptional modulation with type I CRISPR-Cas systems in human cells. Nat Biotechnol 37:1493–1501
pubmed: 31548729
pmcid: 6893126
Pyne ME, Bruder MR, Moo-Young M et al (2016) Harnessing heterologous and endogenous CRISPR-Cas machineries for efficient markerless genome editing in Clostridium. Sci Rep 6:25666
pubmed: 27157668
pmcid: 4860712
Hidalgo-Cantabrana C, Goh YJ, Pan M et al (2019) Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus. Proc Natl Acad Sci U S A 116:15774–15783
pubmed: 31341082
pmcid: 6690032
Zheng Y, Han J, Wang B et al (2019) Characterization and repurposing of the endogenous type I-F CRISPR-Cas system of Zymomonas mobilis for genome engineering. Nucleic Acids Res 47:11461–11475
pubmed: 31647102
pmcid: 6868425
Rath D, Amlinger L, Hoekzema M et al (2015) Efficient programmable gene silencing by Cascade. Nucleic Acids Res 43:237–246
pubmed: 25435544
Gomaa AA, Klumpe HE, Luo ML et al (2014) Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems. MBio 5:e00928–e00913
pubmed: 24473129
pmcid: 3903277
Yosef I, Manor M, Kiro R, Qimron U (2015) Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria. Proc Natl Acad Sci U S A 112:7267–7272
pubmed: 26060300
pmcid: 4466736
Silverman AD, Karim AS, Jewett MC (2020) Cell-free gene expression: an expanded repertoire of applications. Nat Rev Genet 21:151–170
pubmed: 31780816
Liao C, Ttofali F, Slotkowski RA et al (2019) Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis. Nat Commun 10:2948
pubmed: 31270316
pmcid: 6610086
Liao C, Slotkowski RA, Achmedov T, Beisel CL (2019) The Francisella novicida Cas12a is sensitive to the structure downstream of the terminal repeat in CRISPR arrays. RNA Biol 16:404–412
pubmed: 30252595
Marshall R, Beisel CL, Noireaux V (2020) Rapid testing of CRISPR nucleases and guide RNAs in an E. coli cell-free transcription-translation system. STAR Protocols 1:100003
pubmed: 33111065
pmcid: 7580202
Marshall R, Maxwell CS, Collins SP et al (2018) Rapid and scalable characterization of CRISPR technologies using an E. coli cell-free transcription-translation system. Mol Cell 69:146–157.e3
pubmed: 29304331
pmcid: 5976856
Bondy-Denomy J, Pawluk A, Maxwell KL, Davidson AR (2013) Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. Nature 493:429–432
pubmed: 23242138
Davidson AR, Lu W-T, Stanley SY et al (2020) Anti-CRISPRs: protein inhibitors of CRISPR-Cas systems. Annu Rev Biochem 89:309–332
pubmed: 32186918
Wandera KG, Collins SP, Wimmer F et al (2020) An enhanced assay to characterize anti-CRISPR proteins using a cell-free transcription-translation system. Methods 172:42–50
pubmed: 31121300
Watters KE, Fellmann C, Bai HB et al (2018) Systematic discovery of natural CRISPR-Cas12a inhibitors. Science 362:236–239
pubmed: 30190307
pmcid: 6185749
Collias D, Beisel CL (2021) CRISPR technologies and the search for the PAM-free nuclease. Nat Commun 12:555
pubmed: 33483498
pmcid: 7822910
Maxwell CS, Jacobsen T, Marshall R et al (2018) A detailed cell-free transcription-translation-based assay to decipher CRISPR protospacer-adjacent motifs. Methods 143:48–57
pubmed: 29486239
pmcid: 6051895
Leenay RT, Maksimchuk KR, Slotkowski RA et al (2016) Identifying and visualizing functional PAM diversity across CRISPR-Cas systems. Mol Cell 62:137–147
pubmed: 27041224
pmcid: 4826307
Sitaraman K, Esposito D, Klarmann G et al (2004) A novel cell-free protein synthesis system. J Biotechnol 110:257–263
pubmed: 15163516
Marshall R, Maxwell CS, Collins SP et al (2017) Short DNA containing χ sites enhances DNA stability and gene expression in E. coli cell-free transcription-translation systems. Biotechnol Bioeng 114:2137–2141
pubmed: 28475211
pmcid: 5522353
Shin J, Noireaux V (2010) Efficient cell-free expression with the endogenous E. coli RNA polymerase and sigma factor 70. J Biol Eng 4:8
pubmed: 20576148
pmcid: 3161345
Schneider TD, Stephens RM (1990) Sequence logos: a new way to display consensus sequences. Nucleic Acids Res 18:6097–6100
pubmed: 2172928
pmcid: 332411
Collias D, Leenay RT, Slotkowski RA et al (2020) A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9. Sci Adv 6:eabb4054
pubmed: 32832642
pmcid: 7439565
Wimmer F, Mougiakos I, Englert F, Beisel CL (2021) Rapid cell-free characterization of multi-subunit CRISPR effectors and transposons. bioRxiv 2021.10.18.464778; doi: https://doi.org/10.1101/2021.10.18.464778