Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing

Background Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ. Results In th...
Ausführliche Beschreibung

Gespeichert in:

Autor*in:	Guo, Tao [verfasserIn] Feng, Yi-Li Xiao, Jing-Jing Liu, Qian Sun, Xiu-Na Xiang, Ji-Feng Kong, Na Liu, Si-Cheng Chen, Guo-Qiao Wang, Yue Dong, Meng-Meng Cai, Zhen Lin, Hui Cai, Xiu-Jun Xie, An-Yong

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2018

Schlagwörter:	Paired gRNAs CRISPR/Cas9 Accurate NHEJ Templated insertions Precise deletion Knockout Targeted in-frame deletion Genome editing

Anmerkung:	© The Author(s). 2018

Übergeordnetes Werk:	Enthalten in: Genome biology - London : BioMed Central, 2000, 19(2018), 1 vom: 19. Okt.
Übergeordnetes Werk:	volume:19 ; year:2018 ; number:1 ; day:19 ; month:10

Links:	Volltext

DOI / URN:	10.1186/s13059-018-1518-x

Katalog-ID:	SPR030032415

Internformat


LEADER	01000caa a22002652 4500
001	SPR030032415
003	DE-627
005	20230519080340.0
007	cr uuu---uuuuu
008	201007s2018 xx \|\|\|\|\|o 00\| \|\|eng c
024	7		\|a 10.1186/s13059-018-1518-x \|2 doi
035			\|a (DE-627)SPR030032415
035			\|a (SPR)s13059-018-1518-x-e
040			\|a DE-627 \|b ger \|c DE-627 \|e rakwb
041			\|a eng
100	1		\|a Guo, Tao \|e verfasserin \|4 aut
245	1	0	\|a Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing
264		1	\|c 2018
336			\|a Text \|b txt \|2 rdacontent
337			\|a Computermedien \|b c \|2 rdamedia
338			\|a Online-Ressource \|b cr \|2 rdacarrier
500			\|a © The Author(s). 2018
520			\|a Background Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ. Results In the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined “3n”-, “3n + 1”-, or “3n + 2”-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to “3n + 1”-bp, “3n + 2”-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions. Conclusions NHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length.
650		4	\|a Paired gRNAs \|7 (dpeaa)DE-He213
650		4	\|a CRISPR/Cas9 \|7 (dpeaa)DE-He213
650		4	\|a Accurate NHEJ \|7 (dpeaa)DE-He213
650		4	\|a Templated insertions \|7 (dpeaa)DE-He213
650		4	\|a Precise deletion \|7 (dpeaa)DE-He213
650		4	\|a Knockout \|7 (dpeaa)DE-He213
650		4	\|a Targeted in-frame deletion \|7 (dpeaa)DE-He213
650		4	\|a Genome editing \|7 (dpeaa)DE-He213
700	1		\|a Feng, Yi-Li \|4 aut
700	1		\|a Xiao, Jing-Jing \|4 aut
700	1		\|a Liu, Qian \|4 aut
700	1		\|a Sun, Xiu-Na \|4 aut
700	1		\|a Xiang, Ji-Feng \|4 aut
700	1		\|a Kong, Na \|4 aut
700	1		\|a Liu, Si-Cheng \|4 aut
700	1		\|a Chen, Guo-Qiao \|4 aut
700	1		\|a Wang, Yue \|4 aut
700	1		\|a Dong, Meng-Meng \|4 aut
700	1		\|a Cai, Zhen \|4 aut
700	1		\|a Lin, Hui \|4 aut
700	1		\|a Cai, Xiu-Jun \|4 aut
700	1		\|a Xie, An-Yong \|4 aut
773	0	8	\|i Enthalten in \|t Genome biology \|d London : BioMed Central, 2000 \|g 19(2018), 1 vom: 19. Okt. \|w (DE-627)326173617 \|w (DE-600)2040529-7 \|x 1474-760X \|7 nnns
773	1	8	\|g volume:19 \|g year:2018 \|g number:1 \|g day:19 \|g month:10
856	4	0	\|u https://dx.doi.org/10.1186/s13059-018-1518-x \|z kostenfrei \|3 Volltext
912			\|a GBV_USEFLAG_A
912			\|a SYSFLAG_A
912			\|a GBV_SPRINGER
912			\|a SSG-OLC-PHA
912			\|a GBV_ILN_11
912			\|a GBV_ILN_20
912			\|a GBV_ILN_22
912			\|a GBV_ILN_23
912			\|a GBV_ILN_24
912			\|a GBV_ILN_31
912			\|a GBV_ILN_39
912			\|a GBV_ILN_40
912			\|a GBV_ILN_62
912			\|a GBV_ILN_63
912			\|a GBV_ILN_65
912			\|a GBV_ILN_69
912			\|a GBV_ILN_70
912			\|a GBV_ILN_73
912			\|a GBV_ILN_74
912			\|a GBV_ILN_95
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_213
912			\|a GBV_ILN_230
912			\|a GBV_ILN_285
912			\|a GBV_ILN_293
912			\|a GBV_ILN_602
912			\|a GBV_ILN_2003
912			\|a GBV_ILN_2014
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
912			\|a GBV_ILN_4324
912			\|a GBV_ILN_4325
912			\|a GBV_ILN_4338
912			\|a GBV_ILN_4367
912			\|a GBV_ILN_4700
951			\|a AR
952			\|d 19 \|j 2018 \|e 1 \|b 19 \|c 10

Indexfelder

author_variant	t g tg y l f ylf j j x jjx q l ql x n s xns j f x jfx n k nk s c l scl g q c gqc y w yw m m d mmd z c zc h l hl x j c xjc a y x ayx
matchkey_str	article:1474760X:2018----::ansigcuaeohmlguedonnfrfiinpeieeeinnrs
hierarchy_sort_str	2018
publishDate	2018
allfields	10.1186/s13059-018-1518-x doi (DE-627)SPR030032415 (SPR)s13059-018-1518-x-e DE-627 ger DE-627 rakwb eng Guo, Tao verfasserin aut Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2018 Background Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ. Results In the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined “3n”-, “3n + 1”-, or “3n + 2”-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to “3n + 1”-bp, “3n + 2”-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions. Conclusions NHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length. Paired gRNAs (dpeaa)DE-He213 CRISPR/Cas9 (dpeaa)DE-He213 Accurate NHEJ (dpeaa)DE-He213 Templated insertions (dpeaa)DE-He213 Precise deletion (dpeaa)DE-He213 Knockout (dpeaa)DE-He213 Targeted in-frame deletion (dpeaa)DE-He213 Genome editing (dpeaa)DE-He213 Feng, Yi-Li aut Xiao, Jing-Jing aut Liu, Qian aut Sun, Xiu-Na aut Xiang, Ji-Feng aut Kong, Na aut Liu, Si-Cheng aut Chen, Guo-Qiao aut Wang, Yue aut Dong, Meng-Meng aut Cai, Zhen aut Lin, Hui aut Cai, Xiu-Jun aut Xie, An-Yong aut Enthalten in Genome biology London : BioMed Central, 2000 19(2018), 1 vom: 19. Okt. (DE-627)326173617 (DE-600)2040529-7 1474-760X nnns volume:19 year:2018 number:1 day:19 month:10 https://dx.doi.org/10.1186/s13059-018-1518-x kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 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_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 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 19 2018 1 19 10
spelling	10.1186/s13059-018-1518-x doi (DE-627)SPR030032415 (SPR)s13059-018-1518-x-e DE-627 ger DE-627 rakwb eng Guo, Tao verfasserin aut Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2018 Background Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ. Results In the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined “3n”-, “3n + 1”-, or “3n + 2”-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to “3n + 1”-bp, “3n + 2”-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions. Conclusions NHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length. Paired gRNAs (dpeaa)DE-He213 CRISPR/Cas9 (dpeaa)DE-He213 Accurate NHEJ (dpeaa)DE-He213 Templated insertions (dpeaa)DE-He213 Precise deletion (dpeaa)DE-He213 Knockout (dpeaa)DE-He213 Targeted in-frame deletion (dpeaa)DE-He213 Genome editing (dpeaa)DE-He213 Feng, Yi-Li aut Xiao, Jing-Jing aut Liu, Qian aut Sun, Xiu-Na aut Xiang, Ji-Feng aut Kong, Na aut Liu, Si-Cheng aut Chen, Guo-Qiao aut Wang, Yue aut Dong, Meng-Meng aut Cai, Zhen aut Lin, Hui aut Cai, Xiu-Jun aut Xie, An-Yong aut Enthalten in Genome biology London : BioMed Central, 2000 19(2018), 1 vom: 19. Okt. (DE-627)326173617 (DE-600)2040529-7 1474-760X nnns volume:19 year:2018 number:1 day:19 month:10 https://dx.doi.org/10.1186/s13059-018-1518-x kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 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_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 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 19 2018 1 19 10
allfields_unstemmed	10.1186/s13059-018-1518-x doi (DE-627)SPR030032415 (SPR)s13059-018-1518-x-e DE-627 ger DE-627 rakwb eng Guo, Tao verfasserin aut Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2018 Background Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ. Results In the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined “3n”-, “3n + 1”-, or “3n + 2”-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to “3n + 1”-bp, “3n + 2”-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions. Conclusions NHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length. Paired gRNAs (dpeaa)DE-He213 CRISPR/Cas9 (dpeaa)DE-He213 Accurate NHEJ (dpeaa)DE-He213 Templated insertions (dpeaa)DE-He213 Precise deletion (dpeaa)DE-He213 Knockout (dpeaa)DE-He213 Targeted in-frame deletion (dpeaa)DE-He213 Genome editing (dpeaa)DE-He213 Feng, Yi-Li aut Xiao, Jing-Jing aut Liu, Qian aut Sun, Xiu-Na aut Xiang, Ji-Feng aut Kong, Na aut Liu, Si-Cheng aut Chen, Guo-Qiao aut Wang, Yue aut Dong, Meng-Meng aut Cai, Zhen aut Lin, Hui aut Cai, Xiu-Jun aut Xie, An-Yong aut Enthalten in Genome biology London : BioMed Central, 2000 19(2018), 1 vom: 19. Okt. (DE-627)326173617 (DE-600)2040529-7 1474-760X nnns volume:19 year:2018 number:1 day:19 month:10 https://dx.doi.org/10.1186/s13059-018-1518-x kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 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_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 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 19 2018 1 19 10
allfieldsGer	10.1186/s13059-018-1518-x doi (DE-627)SPR030032415 (SPR)s13059-018-1518-x-e DE-627 ger DE-627 rakwb eng Guo, Tao verfasserin aut Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2018 Background Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ. Results In the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined “3n”-, “3n + 1”-, or “3n + 2”-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to “3n + 1”-bp, “3n + 2”-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions. Conclusions NHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length. Paired gRNAs (dpeaa)DE-He213 CRISPR/Cas9 (dpeaa)DE-He213 Accurate NHEJ (dpeaa)DE-He213 Templated insertions (dpeaa)DE-He213 Precise deletion (dpeaa)DE-He213 Knockout (dpeaa)DE-He213 Targeted in-frame deletion (dpeaa)DE-He213 Genome editing (dpeaa)DE-He213 Feng, Yi-Li aut Xiao, Jing-Jing aut Liu, Qian aut Sun, Xiu-Na aut Xiang, Ji-Feng aut Kong, Na aut Liu, Si-Cheng aut Chen, Guo-Qiao aut Wang, Yue aut Dong, Meng-Meng aut Cai, Zhen aut Lin, Hui aut Cai, Xiu-Jun aut Xie, An-Yong aut Enthalten in Genome biology London : BioMed Central, 2000 19(2018), 1 vom: 19. Okt. (DE-627)326173617 (DE-600)2040529-7 1474-760X nnns volume:19 year:2018 number:1 day:19 month:10 https://dx.doi.org/10.1186/s13059-018-1518-x kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 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_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 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 19 2018 1 19 10
allfieldsSound	10.1186/s13059-018-1518-x doi (DE-627)SPR030032415 (SPR)s13059-018-1518-x-e DE-627 ger DE-627 rakwb eng Guo, Tao verfasserin aut Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2018 Background Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ. Results In the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined “3n”-, “3n + 1”-, or “3n + 2”-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to “3n + 1”-bp, “3n + 2”-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions. Conclusions NHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length. Paired gRNAs (dpeaa)DE-He213 CRISPR/Cas9 (dpeaa)DE-He213 Accurate NHEJ (dpeaa)DE-He213 Templated insertions (dpeaa)DE-He213 Precise deletion (dpeaa)DE-He213 Knockout (dpeaa)DE-He213 Targeted in-frame deletion (dpeaa)DE-He213 Genome editing (dpeaa)DE-He213 Feng, Yi-Li aut Xiao, Jing-Jing aut Liu, Qian aut Sun, Xiu-Na aut Xiang, Ji-Feng aut Kong, Na aut Liu, Si-Cheng aut Chen, Guo-Qiao aut Wang, Yue aut Dong, Meng-Meng aut Cai, Zhen aut Lin, Hui aut Cai, Xiu-Jun aut Xie, An-Yong aut Enthalten in Genome biology London : BioMed Central, 2000 19(2018), 1 vom: 19. Okt. (DE-627)326173617 (DE-600)2040529-7 1474-760X nnns volume:19 year:2018 number:1 day:19 month:10 https://dx.doi.org/10.1186/s13059-018-1518-x kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 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_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 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 19 2018 1 19 10
language	English
source	Enthalten in Genome biology 19(2018), 1 vom: 19. Okt. volume:19 year:2018 number:1 day:19 month:10
sourceStr	Enthalten in Genome biology 19(2018), 1 vom: 19. Okt. volume:19 year:2018 number:1 day:19 month:10
format_phy_str_mv	Article
institution	findex.gbv.de
topic_facet	Paired gRNAs CRISPR/Cas9 Accurate NHEJ Templated insertions Precise deletion Knockout Targeted in-frame deletion Genome editing
isfreeaccess_bool	true
container_title	Genome biology
authorswithroles_txt_mv	Guo, Tao @@aut@@ Feng, Yi-Li @@aut@@ Xiao, Jing-Jing @@aut@@ Liu, Qian @@aut@@ Sun, Xiu-Na @@aut@@ Xiang, Ji-Feng @@aut@@ Kong, Na @@aut@@ Liu, Si-Cheng @@aut@@ Chen, Guo-Qiao @@aut@@ Wang, Yue @@aut@@ Dong, Meng-Meng @@aut@@ Cai, Zhen @@aut@@ Lin, Hui @@aut@@ Cai, Xiu-Jun @@aut@@ Xie, An-Yong @@aut@@
publishDateDaySort_date	2018-10-19T00:00:00Z
hierarchy_top_id	326173617
id	SPR030032415
language_de	englisch
fullrecord	<?xml version="1.0" encoding="UTF-8"?><collection xmlns="http://www.loc.gov/MARC21/slim"><record><leader>01000caa a22002652 4500</leader><controlfield tag="001">SPR030032415</controlfield><controlfield tag="003">DE-627</controlfield><controlfield tag="005">20230519080340.0</controlfield><controlfield tag="007">cr uuu---uuuuu</controlfield><controlfield tag="008">201007s2018 xx \|\|\|\|\|o 00\| \|\|eng c</controlfield><datafield tag="024" ind1="7" ind2=" "><subfield code="a">10.1186/s13059-018-1518-x</subfield><subfield code="2">doi</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-627)SPR030032415</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(SPR)s13059-018-1518-x-e</subfield></datafield><datafield tag="040" ind1=" " ind2=" "><subfield code="a">DE-627</subfield><subfield code="b">ger</subfield><subfield code="c">DE-627</subfield><subfield code="e">rakwb</subfield></datafield><datafield tag="041" ind1=" " ind2=" "><subfield code="a">eng</subfield></datafield><datafield tag="100" ind1="1" ind2=" "><subfield code="a">Guo, Tao</subfield><subfield code="e">verfasserin</subfield><subfield code="4">aut</subfield></datafield><datafield tag="245" ind1="1" ind2="0"><subfield code="a">Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing</subfield></datafield><datafield tag="264" ind1=" " ind2="1"><subfield code="c">2018</subfield></datafield><datafield tag="336" ind1=" " ind2=" "><subfield code="a">Text</subfield><subfield code="b">txt</subfield><subfield code="2">rdacontent</subfield></datafield><datafield tag="337" ind1=" " ind2=" "><subfield code="a">Computermedien</subfield><subfield code="b">c</subfield><subfield code="2">rdamedia</subfield></datafield><datafield tag="338" ind1=" " ind2=" "><subfield code="a">Online-Ressource</subfield><subfield code="b">cr</subfield><subfield code="2">rdacarrier</subfield></datafield><datafield tag="500" ind1=" " ind2=" "><subfield code="a">© The Author(s). 2018</subfield></datafield><datafield tag="520" ind1=" " ind2=" "><subfield code="a">Background Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ. Results In the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined “3n”-, “3n + 1”-, or “3n + 2”-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to “3n + 1”-bp, “3n + 2”-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions. Conclusions NHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length.</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Paired gRNAs</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">CRISPR/Cas9</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Accurate NHEJ</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Templated insertions</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Precise deletion</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Knockout</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Targeted in-frame deletion</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Genome editing</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Feng, Yi-Li</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Xiao, Jing-Jing</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Liu, Qian</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Sun, Xiu-Na</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Xiang, Ji-Feng</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Kong, Na</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Liu, Si-Cheng</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Chen, Guo-Qiao</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Wang, Yue</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Dong, Meng-Meng</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Cai, Zhen</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Lin, Hui</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Cai, Xiu-Jun</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Xie, An-Yong</subfield><subfield code="4">aut</subfield></datafield><datafield tag="773" ind1="0" ind2="8"><subfield code="i">Enthalten in</subfield><subfield code="t">Genome biology</subfield><subfield code="d">London : BioMed Central, 2000</subfield><subfield code="g">19(2018), 1 vom: 19. Okt.</subfield><subfield code="w">(DE-627)326173617</subfield><subfield code="w">(DE-600)2040529-7</subfield><subfield code="x">1474-760X</subfield><subfield code="7">nnns</subfield></datafield><datafield tag="773" ind1="1" ind2="8"><subfield code="g">volume:19</subfield><subfield code="g">year:2018</subfield><subfield code="g">number:1</subfield><subfield code="g">day:19</subfield><subfield code="g">month:10</subfield></datafield><datafield tag="856" ind1="4" ind2="0"><subfield code="u">https://dx.doi.org/10.1186/s13059-018-1518-x</subfield><subfield code="z">kostenfrei</subfield><subfield code="3">Volltext</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_USEFLAG_A</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">SYSFLAG_A</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_SPRINGER</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">SSG-OLC-PHA</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_11</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_20</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_22</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_23</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_24</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_31</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_39</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_40</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_62</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_63</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_65</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_69</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_70</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_73</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_74</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_95</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_105</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_110</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_151</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_161</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_170</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_213</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_230</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_285</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_293</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_602</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_2003</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_2014</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4012</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4037</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4112</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4125</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4126</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4249</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4305</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4306</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4307</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4313</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4322</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4323</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4324</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4325</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4338</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4367</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4700</subfield></datafield><datafield tag="951" ind1=" " ind2=" "><subfield code="a">AR</subfield></datafield><datafield tag="952" ind1=" " ind2=" "><subfield code="d">19</subfield><subfield code="j">2018</subfield><subfield code="e">1</subfield><subfield code="b">19</subfield><subfield code="c">10</subfield></datafield></record></collection>
author	Guo, Tao
spellingShingle	Guo, Tao misc Paired gRNAs misc CRISPR/Cas9 misc Accurate NHEJ misc Templated insertions misc Precise deletion misc Knockout misc Targeted in-frame deletion misc Genome editing Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing
authorStr	Guo, Tao
ppnlink_with_tag_str_mv	@@773@@(DE-627)326173617
format	electronic Article
delete_txt_mv	keep
author_role	aut aut aut aut aut aut aut aut aut aut aut aut aut aut aut
collection	springer
remote_str	true
illustrated	Not Illustrated
issn	1474-760X
topic_title	Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing Paired gRNAs (dpeaa)DE-He213 CRISPR/Cas9 (dpeaa)DE-He213 Accurate NHEJ (dpeaa)DE-He213 Templated insertions (dpeaa)DE-He213 Precise deletion (dpeaa)DE-He213 Knockout (dpeaa)DE-He213 Targeted in-frame deletion (dpeaa)DE-He213 Genome editing (dpeaa)DE-He213
topic	misc Paired gRNAs misc CRISPR/Cas9 misc Accurate NHEJ misc Templated insertions misc Precise deletion misc Knockout misc Targeted in-frame deletion misc Genome editing
topic_unstemmed	misc Paired gRNAs misc CRISPR/Cas9 misc Accurate NHEJ misc Templated insertions misc Precise deletion misc Knockout misc Targeted in-frame deletion misc Genome editing
topic_browse	misc Paired gRNAs misc CRISPR/Cas9 misc Accurate NHEJ misc Templated insertions misc Precise deletion misc Knockout misc Targeted in-frame deletion misc Genome editing
format_facet	Elektronische Aufsätze Aufsätze Elektronische Ressource
format_main_str_mv	Text Zeitschrift/Artikel
carriertype_str_mv	cr
hierarchy_parent_title	Genome biology
hierarchy_parent_id	326173617
hierarchy_top_title	Genome biology
isfreeaccess_txt	true
familylinks_str_mv	(DE-627)326173617 (DE-600)2040529-7
title	Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing
ctrlnum	(DE-627)SPR030032415 (SPR)s13059-018-1518-x-e
title_full	Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing
author_sort	Guo, Tao
journal	Genome biology
journalStr	Genome biology
lang_code	eng
isOA_bool	true
recordtype	marc
publishDateSort	2018
contenttype_str_mv	txt
author_browse	Guo, Tao Feng, Yi-Li Xiao, Jing-Jing Liu, Qian Sun, Xiu-Na Xiang, Ji-Feng Kong, Na Liu, Si-Cheng Chen, Guo-Qiao Wang, Yue Dong, Meng-Meng Cai, Zhen Lin, Hui Cai, Xiu-Jun Xie, An-Yong
container_volume	19
format_se	Elektronische Aufsätze
author-letter	Guo, Tao
doi_str_mv	10.1186/s13059-018-1518-x
title_sort	harnessing accurate non-homologous end joining for efficient precise deletion in crispr/cas9-mediated genome editing
title_auth	Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing
abstract	Background Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ. Results In the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined “3n”-, “3n + 1”-, or “3n + 2”-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to “3n + 1”-bp, “3n + 2”-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions. Conclusions NHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length. © The Author(s). 2018
abstractGer	Background Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ. Results In the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined “3n”-, “3n + 1”-, or “3n + 2”-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to “3n + 1”-bp, “3n + 2”-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions. Conclusions NHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length. © The Author(s). 2018
abstract_unstemmed	Background Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ. Results In the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined “3n”-, “3n + 1”-, or “3n + 2”-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to “3n + 1”-bp, “3n + 2”-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions. Conclusions NHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length. © The Author(s). 2018
collection_details	GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 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_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 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	1
title_short	Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing
url	https://dx.doi.org/10.1186/s13059-018-1518-x
remote_bool	true
author2	Feng, Yi-Li Xiao, Jing-Jing Liu, Qian Sun, Xiu-Na Xiang, Ji-Feng Kong, Na Liu, Si-Cheng Chen, Guo-Qiao Wang, Yue Dong, Meng-Meng Cai, Zhen Lin, Hui Cai, Xiu-Jun Xie, An-Yong
author2Str	Feng, Yi-Li Xiao, Jing-Jing Liu, Qian Sun, Xiu-Na Xiang, Ji-Feng Kong, Na Liu, Si-Cheng Chen, Guo-Qiao Wang, Yue Dong, Meng-Meng Cai, Zhen Lin, Hui Cai, Xiu-Jun Xie, An-Yong
ppnlink	326173617
mediatype_str_mv	c
isOA_txt	true
hochschulschrift_bool	false
doi_str	10.1186/s13059-018-1518-x
up_date	2024-07-03T13:27:38.795Z
_version_	1803564622859468800
fullrecord_marcxml	<?xml version="1.0" encoding="UTF-8"?><collection xmlns="http://www.loc.gov/MARC21/slim"><record><leader>01000caa a22002652 4500</leader><controlfield tag="001">SPR030032415</controlfield><controlfield tag="003">DE-627</controlfield><controlfield tag="005">20230519080340.0</controlfield><controlfield tag="007">cr uuu---uuuuu</controlfield><controlfield tag="008">201007s2018 xx \|\|\|\|\|o 00\| \|\|eng c</controlfield><datafield tag="024" ind1="7" ind2=" "><subfield code="a">10.1186/s13059-018-1518-x</subfield><subfield code="2">doi</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-627)SPR030032415</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(SPR)s13059-018-1518-x-e</subfield></datafield><datafield tag="040" ind1=" " ind2=" "><subfield code="a">DE-627</subfield><subfield code="b">ger</subfield><subfield code="c">DE-627</subfield><subfield code="e">rakwb</subfield></datafield><datafield tag="041" ind1=" " ind2=" "><subfield code="a">eng</subfield></datafield><datafield tag="100" ind1="1" ind2=" "><subfield code="a">Guo, Tao</subfield><subfield code="e">verfasserin</subfield><subfield code="4">aut</subfield></datafield><datafield tag="245" ind1="1" ind2="0"><subfield code="a">Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing</subfield></datafield><datafield tag="264" ind1=" " ind2="1"><subfield code="c">2018</subfield></datafield><datafield tag="336" ind1=" " ind2=" "><subfield code="a">Text</subfield><subfield code="b">txt</subfield><subfield code="2">rdacontent</subfield></datafield><datafield tag="337" ind1=" " ind2=" "><subfield code="a">Computermedien</subfield><subfield code="b">c</subfield><subfield code="2">rdamedia</subfield></datafield><datafield tag="338" ind1=" " ind2=" "><subfield code="a">Online-Ressource</subfield><subfield code="b">cr</subfield><subfield code="2">rdacarrier</subfield></datafield><datafield tag="500" ind1=" " ind2=" "><subfield code="a">© The Author(s). 2018</subfield></datafield><datafield tag="520" ind1=" " ind2=" "><subfield code="a">Background Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ. Results In the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined “3n”-, “3n + 1”-, or “3n + 2”-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to “3n + 1”-bp, “3n + 2”-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions. Conclusions NHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length.</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Paired gRNAs</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">CRISPR/Cas9</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Accurate NHEJ</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Templated insertions</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Precise deletion</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Knockout</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Targeted in-frame deletion</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Genome editing</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Feng, Yi-Li</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Xiao, Jing-Jing</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Liu, Qian</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Sun, Xiu-Na</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Xiang, Ji-Feng</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Kong, Na</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Liu, Si-Cheng</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Chen, Guo-Qiao</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Wang, Yue</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Dong, Meng-Meng</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Cai, Zhen</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Lin, Hui</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Cai, Xiu-Jun</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Xie, An-Yong</subfield><subfield code="4">aut</subfield></datafield><datafield tag="773" ind1="0" ind2="8"><subfield code="i">Enthalten in</subfield><subfield code="t">Genome biology</subfield><subfield code="d">London : BioMed Central, 2000</subfield><subfield code="g">19(2018), 1 vom: 19. Okt.</subfield><subfield code="w">(DE-627)326173617</subfield><subfield code="w">(DE-600)2040529-7</subfield><subfield code="x">1474-760X</subfield><subfield code="7">nnns</subfield></datafield><datafield tag="773" ind1="1" ind2="8"><subfield code="g">volume:19</subfield><subfield code="g">year:2018</subfield><subfield code="g">number:1</subfield><subfield code="g">day:19</subfield><subfield code="g">month:10</subfield></datafield><datafield tag="856" ind1="4" ind2="0"><subfield code="u">https://dx.doi.org/10.1186/s13059-018-1518-x</subfield><subfield code="z">kostenfrei</subfield><subfield code="3">Volltext</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_USEFLAG_A</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">SYSFLAG_A</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_SPRINGER</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">SSG-OLC-PHA</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_11</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_20</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_22</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_23</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_24</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_31</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_39</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_40</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_62</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_63</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_65</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_69</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_70</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_73</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_74</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_95</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_105</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_110</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_151</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_161</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_170</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_213</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_230</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_285</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_293</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_602</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_2003</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_2014</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4012</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4037</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4112</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4125</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4126</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4249</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4305</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4306</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4307</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4313</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4322</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4323</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4324</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4325</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4338</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4367</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ILN_4700</subfield></datafield><datafield tag="951" ind1=" " ind2=" "><subfield code="a">AR</subfield></datafield><datafield tag="952" ind1=" " ind2=" "><subfield code="d">19</subfield><subfield code="j">2018</subfield><subfield code="e">1</subfield><subfield code="b">19</subfield><subfield code="c">10</subfield></datafield></record></collection>
score	7.3993244

Nicht das Richtige dabei?

Schreiben Sie uns!

Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing

Nicht das Richtige dabei?

Zugang & Verfügbarkeit

Vorhandene Bände

Nicht das Richtige dabei?