Having a Same Type IIS Enzyme’s Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis

Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRI...
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Gespeichert in:

Autor*in:	M. Moniruzzaman [verfasserIn] Yun Zhong [verfasserIn] Zhifeng Huang [verfasserIn] Guangyan Zhong [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2022

Schlagwörter:	gRNA designing modular cloning restriction digestion genome editing base editing plant transformation

Übergeordnetes Werk:	In: International Journal of Molecular Sciences - MDPI AG, 2003, 23(2022), 9, p 4889
Übergeordnetes Werk:	volume:23 ; year:2022 ; number:9, p 4889

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc Journal toc

DOI / URN:	10.3390/ijms23094889

Katalog-ID:	DOAJ031149812

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520			\|a Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a <i<Cas12a</i< nuclease and four gRNAs was used for <i<Agrobacterium</i<-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors.
650		4	\|a gRNA designing
650		4	\|a modular cloning
650		4	\|a restriction digestion
650		4	\|a genome editing
650		4	\|a base editing
650		4	\|a plant transformation
653		0	\|a Biology (General)
653		0	\|a Chemistry
700	0		\|a Yun Zhong \|e verfasserin \|4 aut
700	0		\|a Zhifeng Huang \|e verfasserin \|4 aut
700	0		\|a Guangyan Zhong \|e verfasserin \|4 aut
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912			\|a GBV_ILN_39
912			\|a GBV_ILN_40
912			\|a GBV_ILN_60
912			\|a GBV_ILN_62
912			\|a GBV_ILN_63
912			\|a GBV_ILN_65
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912			\|a GBV_ILN_70
912			\|a GBV_ILN_73
912			\|a GBV_ILN_74
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912			\|a GBV_ILN_105
912			\|a GBV_ILN_110
912			\|a GBV_ILN_151
912			\|a GBV_ILN_161
912			\|a GBV_ILN_170
912			\|a GBV_ILN_206
912			\|a GBV_ILN_213
912			\|a GBV_ILN_224
912			\|a GBV_ILN_230
912			\|a GBV_ILN_285
912			\|a GBV_ILN_293
912			\|a GBV_ILN_602
912			\|a GBV_ILN_2005
912			\|a GBV_ILN_2009
912			\|a GBV_ILN_2011
912			\|a GBV_ILN_2014
912			\|a GBV_ILN_2055
912			\|a GBV_ILN_2111
912			\|a GBV_ILN_4012
912			\|a GBV_ILN_4037
912			\|a GBV_ILN_4112
912			\|a GBV_ILN_4125
912			\|a GBV_ILN_4126
912			\|a GBV_ILN_4249
912			\|a GBV_ILN_4305
912			\|a GBV_ILN_4306
912			\|a GBV_ILN_4307
912			\|a GBV_ILN_4313
912			\|a GBV_ILN_4322
912			\|a GBV_ILN_4323
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951			\|a AR
952			\|d 23 \|j 2022 \|e 9, p 4889

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allfields	10.3390/ijms23094889 doi (DE-627)DOAJ031149812 (DE-599)DOAJ0711992b98ac4d0c9db13a942f608a54 DE-627 ger DE-627 rakwb eng QH301-705.5 QD1-999 M. Moniruzzaman verfasserin aut Having a Same Type IIS Enzyme’s Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a <i<Cas12a</i< nuclease and four gRNAs was used for <i<Agrobacterium</i<-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors. gRNA designing modular cloning restriction digestion genome editing base editing plant transformation Biology (General) Chemistry Yun Zhong verfasserin aut Zhifeng Huang verfasserin aut Guangyan Zhong verfasserin aut In International Journal of Molecular Sciences MDPI AG, 2003 23(2022), 9, p 4889 (DE-627)316340715 (DE-600)2019364-6 14220067 nnns volume:23 year:2022 number:9, p 4889 https://doi.org/10.3390/ijms23094889 kostenfrei https://doaj.org/article/0711992b98ac4d0c9db13a942f608a54 kostenfrei https://www.mdpi.com/1422-0067/23/9/4889 kostenfrei https://doaj.org/toc/1661-6596 Journal toc kostenfrei https://doaj.org/toc/1422-0067 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 23 2022 9, p 4889
spelling	10.3390/ijms23094889 doi (DE-627)DOAJ031149812 (DE-599)DOAJ0711992b98ac4d0c9db13a942f608a54 DE-627 ger DE-627 rakwb eng QH301-705.5 QD1-999 M. Moniruzzaman verfasserin aut Having a Same Type IIS Enzyme’s Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a <i<Cas12a</i< nuclease and four gRNAs was used for <i<Agrobacterium</i<-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors. gRNA designing modular cloning restriction digestion genome editing base editing plant transformation Biology (General) Chemistry Yun Zhong verfasserin aut Zhifeng Huang verfasserin aut Guangyan Zhong verfasserin aut In International Journal of Molecular Sciences MDPI AG, 2003 23(2022), 9, p 4889 (DE-627)316340715 (DE-600)2019364-6 14220067 nnns volume:23 year:2022 number:9, p 4889 https://doi.org/10.3390/ijms23094889 kostenfrei https://doaj.org/article/0711992b98ac4d0c9db13a942f608a54 kostenfrei https://www.mdpi.com/1422-0067/23/9/4889 kostenfrei https://doaj.org/toc/1661-6596 Journal toc kostenfrei https://doaj.org/toc/1422-0067 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 23 2022 9, p 4889
allfields_unstemmed	10.3390/ijms23094889 doi (DE-627)DOAJ031149812 (DE-599)DOAJ0711992b98ac4d0c9db13a942f608a54 DE-627 ger DE-627 rakwb eng QH301-705.5 QD1-999 M. Moniruzzaman verfasserin aut Having a Same Type IIS Enzyme’s Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a <i<Cas12a</i< nuclease and four gRNAs was used for <i<Agrobacterium</i<-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors. gRNA designing modular cloning restriction digestion genome editing base editing plant transformation Biology (General) Chemistry Yun Zhong verfasserin aut Zhifeng Huang verfasserin aut Guangyan Zhong verfasserin aut In International Journal of Molecular Sciences MDPI AG, 2003 23(2022), 9, p 4889 (DE-627)316340715 (DE-600)2019364-6 14220067 nnns volume:23 year:2022 number:9, p 4889 https://doi.org/10.3390/ijms23094889 kostenfrei https://doaj.org/article/0711992b98ac4d0c9db13a942f608a54 kostenfrei https://www.mdpi.com/1422-0067/23/9/4889 kostenfrei https://doaj.org/toc/1661-6596 Journal toc kostenfrei https://doaj.org/toc/1422-0067 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 23 2022 9, p 4889
allfieldsGer	10.3390/ijms23094889 doi (DE-627)DOAJ031149812 (DE-599)DOAJ0711992b98ac4d0c9db13a942f608a54 DE-627 ger DE-627 rakwb eng QH301-705.5 QD1-999 M. Moniruzzaman verfasserin aut Having a Same Type IIS Enzyme’s Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a <i<Cas12a</i< nuclease and four gRNAs was used for <i<Agrobacterium</i<-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors. gRNA designing modular cloning restriction digestion genome editing base editing plant transformation Biology (General) Chemistry Yun Zhong verfasserin aut Zhifeng Huang verfasserin aut Guangyan Zhong verfasserin aut In International Journal of Molecular Sciences MDPI AG, 2003 23(2022), 9, p 4889 (DE-627)316340715 (DE-600)2019364-6 14220067 nnns volume:23 year:2022 number:9, p 4889 https://doi.org/10.3390/ijms23094889 kostenfrei https://doaj.org/article/0711992b98ac4d0c9db13a942f608a54 kostenfrei https://www.mdpi.com/1422-0067/23/9/4889 kostenfrei https://doaj.org/toc/1661-6596 Journal toc kostenfrei https://doaj.org/toc/1422-0067 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 23 2022 9, p 4889
allfieldsSound	10.3390/ijms23094889 doi (DE-627)DOAJ031149812 (DE-599)DOAJ0711992b98ac4d0c9db13a942f608a54 DE-627 ger DE-627 rakwb eng QH301-705.5 QD1-999 M. Moniruzzaman verfasserin aut Having a Same Type IIS Enzyme’s Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a <i<Cas12a</i< nuclease and four gRNAs was used for <i<Agrobacterium</i<-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors. gRNA designing modular cloning restriction digestion genome editing base editing plant transformation Biology (General) Chemistry Yun Zhong verfasserin aut Zhifeng Huang verfasserin aut Guangyan Zhong verfasserin aut In International Journal of Molecular Sciences MDPI AG, 2003 23(2022), 9, p 4889 (DE-627)316340715 (DE-600)2019364-6 14220067 nnns volume:23 year:2022 number:9, p 4889 https://doi.org/10.3390/ijms23094889 kostenfrei https://doaj.org/article/0711992b98ac4d0c9db13a942f608a54 kostenfrei https://www.mdpi.com/1422-0067/23/9/4889 kostenfrei https://doaj.org/toc/1661-6596 Journal toc kostenfrei https://doaj.org/toc/1422-0067 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 23 2022 9, p 4889
language	English
source	In International Journal of Molecular Sciences 23(2022), 9, p 4889 volume:23 year:2022 number:9, p 4889
sourceStr	In International Journal of Molecular Sciences 23(2022), 9, p 4889 volume:23 year:2022 number:9, p 4889
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authorswithroles_txt_mv	M. Moniruzzaman @@aut@@ Yun Zhong @@aut@@ Zhifeng Huang @@aut@@ Guangyan Zhong @@aut@@
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callnumber-first	Q - Science
author	M. Moniruzzaman
spellingShingle	M. Moniruzzaman misc QH301-705.5 misc QD1-999 misc gRNA designing misc modular cloning misc restriction digestion misc genome editing misc base editing misc plant transformation misc Biology (General) misc Chemistry Having a Same Type IIS Enzyme’s Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis
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topic_title	QH301-705.5 QD1-999 Having a Same Type IIS Enzyme’s Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis gRNA designing modular cloning restriction digestion genome editing base editing plant transformation
topic	misc QH301-705.5 misc QD1-999 misc gRNA designing misc modular cloning misc restriction digestion misc genome editing misc base editing misc plant transformation misc Biology (General) misc Chemistry
topic_unstemmed	misc QH301-705.5 misc QD1-999 misc gRNA designing misc modular cloning misc restriction digestion misc genome editing misc base editing misc plant transformation misc Biology (General) misc Chemistry
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title	Having a Same Type IIS Enzyme’s Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis
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title_full	Having a Same Type IIS Enzyme’s Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis
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author_browse	M. Moniruzzaman Yun Zhong Zhifeng Huang Guangyan Zhong
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title_sort	having a same type iis enzyme’s restriction site on guide rna sequence does not affect golden gate (gg) cloning and subsequent crispr/cas mutagenesis
callnumber	QH301-705.5
title_auth	Having a Same Type IIS Enzyme’s Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis
abstract	Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a <i<Cas12a</i< nuclease and four gRNAs was used for <i<Agrobacterium</i<-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors.
abstractGer	Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a <i<Cas12a</i< nuclease and four gRNAs was used for <i<Agrobacterium</i<-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors.
abstract_unstemmed	Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a <i<Cas12a</i< nuclease and four gRNAs was used for <i<Agrobacterium</i<-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors.
collection_details	GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700
container_issue	9, p 4889
title_short	Having a Same Type IIS Enzyme’s Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis
url	https://doi.org/10.3390/ijms23094889 https://doaj.org/article/0711992b98ac4d0c9db13a942f608a54 https://www.mdpi.com/1422-0067/23/9/4889 https://doaj.org/toc/1661-6596 https://doaj.org/toc/1422-0067
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author2	Yun Zhong Zhifeng Huang Guangyan Zhong
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up_date	2024-07-03T19:00:58.544Z
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Having a Same Type IIS Enzyme’s Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis

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