Precise DNA Cleavage Using CRISPR-SpRYgests

Overview

Journal Nat Biotechnol

Specialty Biotechnology

Date 2022 Oct 6

PMID 36203014

Authors

Kathleen A Christie

Jimmy A Guo

Rachel A Silverstein

Roman M Doll

Megumu Mabuchi

Hannah E Stutzman

Jiecong Lin

Linyuan Ma

Russell T Walton

Luca Pinello

G Brett Robb

Benjamin P Kleinstiver

Affiliations

Soon will be listed here.

Abstract

Methods for in vitro DNA cleavage and molecular cloning remain unable to precisely cleave DNA directly adjacent to bases of interest. Restriction enzymes (REs) must bind specific motifs, whereas wild-type CRISPR-Cas9 or CRISPR-Cas12 nucleases require protospacer adjacent motifs (PAMs). Here we explore the utility of our previously reported near-PAMless SpCas9 variant, named SpRY, to serve as a universal DNA cleavage tool for various cloning applications. By performing SpRY DNA digests (SpRYgests) using more than 130 guide RNAs (gRNAs) sampling a wide diversity of PAMs, we discovered that SpRY is PAMless in vitro and can cleave DNA at practically any sequence, including sites refractory to cleavage with wild-type SpCas9. We illustrate the versatility and effectiveness of SpRYgests to improve the precision of several cloning workflows, including those not possible with REs or canonical CRISPR nucleases. We also optimize a rapid and simple one-pot gRNA synthesis protocol to streamline SpRYgest implementation. Together, SpRYgests can improve various DNA engineering applications that benefit from precise DNA breaks.

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References

Loenen W, Dryden D, Raleigh E, Wilson G, Murray N . Highlights of the DNA cutters: a short history of the restriction enzymes. Nucleic Acids Res. 2013; 42(1):3-19. PMC: 3874209. DOI: 10.1093/nar/gkt990. View

Gibson D, Young L, Chuang R, Venter J, Hutchison 3rd C, Smith H . Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009; 6(5):343-5. DOI: 10.1038/nmeth.1318. View

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna J, Charpentier E . A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337(6096):816-21. PMC: 6286148. DOI: 10.1126/science.1225829. View

Collias D, Beisel C . CRISPR technologies and the search for the PAM-free nuclease. Nat Commun. 2021; 12(1):555. PMC: 7822910. DOI: 10.1038/s41467-020-20633-y. View

Wang J, Wang A, Li K, Wang B, Jin S, Reiser M . CRISPR/Cas9 nuclease cleavage combined with Gibson assembly for seamless cloning. Biotechniques. 2015; 58(4):161-70. DOI: 10.2144/000114261. View

Jiang W, Zhao X, Gabrieli T, Lou C, Ebenstein Y, Zhu T . Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters. Nat Commun. 2015; 6:8101. PMC: 4569707. DOI: 10.1038/ncomms9101. View

Jiang W, Zhu T . Targeted isolation and cloning of 100-kb microbial genomic sequences by Cas9-assisted targeting of chromosome segments. Nat Protoc. 2016; 11(5):960-75. DOI: 10.1038/nprot.2016.055. View

Li S, Zhao G, Wang J . C-Brick: A New Standard for Assembly of Biological Parts Using Cpf1. ACS Synth Biol. 2016; 5(12):1383-1388. DOI: 10.1021/acssynbio.6b00114. View

Jeong Y, Yu J, Bae S . Construction of non-canonical PAM-targeting adenosine base editors by restriction enzyme-free DNA cloning using CRISPR-Cas9. Sci Rep. 2019; 9(1):4939. PMC: 6426851. DOI: 10.1038/s41598-019-41356-1. View

10.

Shola D, Yang C, Kewaldar V, Kar P, Bustos V . New Additions to the CRISPR Toolbox: CRISPR- and CRISPR- for Donor Construction in Genome Editing. CRISPR J. 2020; 3(2):109-122. PMC: 7194329. DOI: 10.1089/crispr.2019.0062. View

11.

Walton R, Christie K, Whittaker M, Kleinstiver B . Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science. 2020; 368(6488):290-296. PMC: 7297043. DOI: 10.1126/science.aba8853. View

12.

Jiang W, Bikard D, Cox D, Zhang F, Marraffini L . RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 2013; 31(3):233-9. PMC: 3748948. DOI: 10.1038/nbt.2508. View

13.

Kleinstiver B, Prew M, Tsai S, Topkar V, Nguyen N, Zheng Z . Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015; 523(7561):481-5. PMC: 4540238. DOI: 10.1038/nature14592. View

14.

Doench J, Hartenian E, Graham D, Tothova Z, Hegde M, Smith I . Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol. 2014; 32(12):1262-7. PMC: 4262738. DOI: 10.1038/nbt.3026. View

15.

Lundberg S, Erion G, Chen H, DeGrave A, Prutkin J, Nair B . From Local Explanations to Global Understanding with Explainable AI for Trees. Nat Mach Intell. 2020; 2(1):56-67. PMC: 7326367. DOI: 10.1038/s42256-019-0138-9. View

16.

Thyme S, Akhmetova L, Montague T, Valen E, Schier A . Internal guide RNA interactions interfere with Cas9-mediated cleavage. Nat Commun. 2016; 7:11750. PMC: 4906408. DOI: 10.1038/ncomms11750. View

17.

Moreb E, Lynch M . A Meta-Analysis of gRNA Library Screens Enables an Improved Understanding of the Impact of gRNA Folding and Structural Stability on CRISPR-Cas9 Activity. CRISPR J. 2022; 5(1):146-154. DOI: 10.1089/crispr.2021.0084. View

18.

Swarts D, Jore M, Westra E, Zhu Y, Janssen J, Snijders A . DNA-guided DNA interference by a prokaryotic Argonaute. Nature. 2014; 507(7491):258-261. PMC: 4697943. DOI: 10.1038/nature12971. View

19.

Enghiad B, Zhao H . Programmable DNA-Guided Artificial Restriction Enzymes. ACS Synth Biol. 2017; 6(5):752-757. DOI: 10.1021/acssynbio.6b00324. View

20.

Enghiad B, Xue P, Singh N, Boob A, Shi C, Petrov V . PlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction. Nat Commun. 2022; 13(1):2697. PMC: 9110713. DOI: 10.1038/s41467-022-30355-y. View