Abstract
A PCR-independent in vitro site-directed mutagenesis method was established. Cas12a from Francisella novicida (FnCas12a) linearizes the plasmid with single digestion. T5 exonuclease removes the target nucleotide. A short single- or double-stranded mutagenic oligonucleotide introduces the mutation. This rapid and simple mutagenesis method is referred to as FnCas12a and T5 exonuclease mediated site-directed mutagenesis system (CT5-SDM). The platform is also suitable for the mutagenesis of plasmids larger than 10 kb.
Key Points
-
Site-directed mutagenesis mediated by single-stranded DNA.
-
Removing target site with T5 exonuclease.
-
Highly efficient cleavage of target DNA with FnCas12a.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
AlMalki FA, Flemming CS, Zhang J, Feng M, Sedelnikova SE, Ceska T, Rafferty JB, Sayers JR, Artymiuk PJ (2016) Direct observation of DNA threading in flap endonuclease complexes. Nat Struct Mol Biol 23:640–646. https://doi.org/10.1038/nsmb.3241
Ceska TA, Sayers JR, Stier G, Suck D (1996) A helical arch allowing single-stranded DNA to thread through T5 5'-exonuclease. Nature 382:90–93. https://doi.org/10.1038/382090a0
Chow RD, Wang G, Ye L, Codina A, Kim HR, Shen L, Dong MB, Errami Y, Chen S (2019) In vivo profiling of metastatic double knockouts through CRISPR-Cpf1 screens. Nat Methods 16:405–408. https://doi.org/10.1038/s41592-019-0371-5
Ellis HM, Yu D, DiTizio T, Court DL (2001) High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc Natl Acad Sci U S A 98:6742–6746. https://doi.org/10.1073/pnas.121164898
Endo A, Masafumi M, Kaya H, Toki S (2016) Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida. Sci Rep 6:38169. https://doi.org/10.1038/srep38169
Gasiunas G, Barrangou R, Horvath P, Siksnys V (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A 109:E2579–E2586. https://doi.org/10.1073/pnas.1208507109
Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345. https://doi.org/10.1038/nmeth.1318
Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59. https://doi.org/10.1016/0378-1119(89)90358-2
Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327:167–170. https://doi.org/10.1126/science.1179555
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821. https://doi.org/10.1126/science.1225829
Kim Y, Cheong SA, Lee JG, Lee SW, Lee MS, Baek IJ, Sung YH (2016) Generation of knockout mice by Cpf1-mediated gene targeting. Nat Biotechnol 34:808–810. https://doi.org/10.1038/nbt.3614
Lei C, Li SY, Liu JK, Zheng X, Zhao GP, Wang J (2017) The CCTL (Cpf1-assisted cutting and Taq DNA ligase-assisted ligation) method for efficient editing of large DNA constructs in vitro. Nucleic Acids Res 45:e74. https://doi.org/10.1093/nar/gkx018
Li MZ, Elledge SJ (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4:251–256. https://doi.org/10.1038/nmeth1010
Li SY, Zhao GP, Wang J (2016) C-Brick: a new standard for assembly of biological parts using Cpf1. ACS Synth Biol 5:1383–1388. https://doi.org/10.1021/acssynbio.6b00114
Lovett ST, Hurley RL, Sutera VA Jr, Aubuchon RH, Lebedeva MA (2002) Crossing over between regions of limited homology in Escherichia coli. RecA-dependent and RecA-independent pathways. Genetics 160:851–859
Marini F 3rd, Naeem A, Lapeyre JN (1993) An efficient 1-tube PCR method for internal site-directed mutagenesis of large amplified molecules. Nucleic Acids Res 21:2277–2278. https://doi.org/10.1093/nar/21.9.2277
Mojica FJ, Diez-Villasenor C, Soria E, Juez G (2000) Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol Microbiol 36:244–246. https://doi.org/10.1046/j.1365-2958.2000.01838.x
Picard V, Ersdal-Badju E, Lu A, Bock SC (1994) A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA polymerase. Nucleic Acids Res 22:2587–2591. https://doi.org/10.1093/nar/22.13.2587
Sansbury BM, Wagner AM, Tarcic G, Barth S, Nitzan E, Goldfus R, Vidne M, Kmiec EB (2019) CRISPR-directed gene editing catalyzes precise gene segment replacement in vitro enabling a novel method for multiplex site-directed mutagenesis. Crispr J 2:121–132. https://doi.org/10.1089/crispr.2018.0054
Schunder E, Rydzewski K, Grunow R, Heuner K (2013) First indication for a functional CRISPR/Cas system in Francisella tularensis. Int J Med Microbiol 303:51–60. https://doi.org/10.1016/j.ijmm.2012.11.004
Sharan SK, Thomason LC, Kuznetsov SG, Court DL (2009) Recombineering: a homologous recombination-based method of genetic engineering. Nat Protoc 4:206–223. https://doi.org/10.1038/nprot.2008.227
She W, Ni J, Shui K, Wang F, He R, Xue J, Reetz MT, Li A, Ma L (2018) Rapid and error-free site-directed mutagenesis by a PCR-free in vitro CRISPR/Cas9-mediated mutagenic system. ACS Synth Biol 7:2236–2244. https://doi.org/10.1021/acssynbio.8b00245
Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Sorek R, Lawrence CM, Wiedenheft B (2013) CRISPR-mediated adaptive immune systems in bacteria and archaea. Annu Rev Biochem 82:237–266. https://doi.org/10.1146/annurev-biochem-072911-172315
Swiat MA, Dashko S, den Ridder M, Wijsman M, van der Oost J, Daran JM, Daran-Lapujade P (2017) FnCpf1: a novel and efficient genome editing tool for Saccharomyces cerevisiae. Nucleic Acids Res 45:12585–12598. https://doi.org/10.1093/nar/gkx1007
Wiedenheft B, Sternberg SH, Doudna JA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482:331–338. https://doi.org/10.1038/nature10886
Xia Y, Li K, Li J, Wang T, Gu L, Xun L (2019) T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis. Nucleic Acids Res 47:e15. https://doi.org/10.1093/nar/gky1169
Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:759–771. https://doi.org/10.1016/j.cell.2015.09.038
Zhang Y, Long C, Li H, McAnally JR, Baskin KK, Shelton JM, Bassel-Duby R, Olson EN (2017) CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice. Sci Adv 3:e1602814. https://doi.org/10.1126/sciadv.1602814
Zhang Y, Werling U, Edelmann W (2012) SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucleic Acids Res 40:e55. https://doi.org/10.1093/nar/gkr1288
Zheng L, Baumann U, Reymond JL (2004) An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res 32:e115. https://doi.org/10.1093/nar/gnh110
Zhu B, Cai G, Hall EO, Freeman GJ (2007) In-Fusion™ assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. BioTechniques 43:354–359. https://doi.org/10.2144/000112536
Funding
This work was supported by the National Natural Science Foundation of China (21977026 and 21702052).
Author information
Authors and Affiliations
Contributions
LM and CZ conceived and designed the study; MD carried out almost all experiments. MD, CZ, and FW performed the analysis of all the data; MD and QL were responsible for all the figures; FW, RH, and QL were involved with plasmid construction and FnCas12a expression; CZ wrote the manuscript; CZ and AL revised the manuscript. All authors read and approved the manuscript.
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
ESM 1
(PDF 2178 kb)
Rights and permissions
About this article
Cite this article
Dong, M., Wang, F., Li, Q. et al. A single digestion, single-stranded oligonucleotide mediated PCR-independent site-directed mutagenesis method. Appl Microbiol Biotechnol 104, 3993–4003 (2020). https://doi.org/10.1007/s00253-020-10477-3
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-020-10477-3