Dear Editor, Recently, engineered endonucleases, such as Zinc-Finger Nucleases （ZFNs） （Carroll, 2011）, Transcription Activator-Like Effector Nucleases （TALENs） （Mahfouz et al., 2011; Li et al., 2012）, and Clu...Dear Editor, Recently, engineered endonucleases, such as Zinc-Finger Nucleases （ZFNs） （Carroll, 2011）, Transcription Activator-Like Effector Nucleases （TALENs） （Mahfouz et al., 2011; Li et al., 2012）, and Clustered Regularly Interspaced Short Palindromic Repeats （CRISPR）-associated （Cas） systems （Cong et al., 2013） have been successfully used for gene editing in a variety of species. These systems generate double-strand breaks （DSBs） at target loci to drive site-specific DNA sequence modifica- tions. The modifications include sequence insertion and deletion and other mutations in the host genomes via the error-prone non-homologous end joining （NHEJ） pathway or sequence correction or replacement through the error-free homologous recombination （HR） pathway （Symington and Gautier, 2011）. Here, we show that the CRISPR-Cas system can be applied to generate targeted gene mutations and gene corrections in plants, and the system can also be readily engi- neered to achieve deletion of large DNA fragments and for multiplex gene editing in plants.展开更多
The complete genome of Marek's disease virus (MDV) strain GX0101, which was integrated with the LTR sequences of REV, was cloned in Escherichia coli as a bacterial artificial chromosome (BAC). BAC vector sequences...The complete genome of Marek's disease virus (MDV) strain GX0101, which was integrated with the LTR sequences of REV, was cloned in Escherichia coli as a bacterial artificial chromosome (BAC). BAC vector sequences were introduced into the US2 locus of the MDV genome by homologous recombination. The viral DNA containing the BAC vector was used to transform Escherichia coli strain of DH10B. Then the recombinant virus was successfully rescued by transfection of the recombinant BAC DNA into primary chicken embryo fibroblast (CEF). This BAC viral clone was named bac-GX0101. When the reconstituted virus was inoculated into 1-day-old birds, visceral tumors could be detected as early as 62 d post infection. There was no difference in growth ability and pathogenicity to birds between the BAC derived virus and its parental virus. The BAC derived virus maintained its oncogenicity and immunosuppressive effects. In conclusion, the complete genome of GX0101 strain was successfully cloned into BAC and the infectious clone was rescued. With the powerful BAC manipulation system, the infectious clone will provide a useful tool for further understanding the functional roles of the inserted REV-LTR sequence in the GX0101 strain of MDV.展开更多
文摘Dear Editor, Recently, engineered endonucleases, such as Zinc-Finger Nucleases （ZFNs） （Carroll, 2011）, Transcription Activator-Like Effector Nucleases （TALENs） （Mahfouz et al., 2011; Li et al., 2012）, and Clustered Regularly Interspaced Short Palindromic Repeats （CRISPR）-associated （Cas） systems （Cong et al., 2013） have been successfully used for gene editing in a variety of species. These systems generate double-strand breaks （DSBs） at target loci to drive site-specific DNA sequence modifica- tions. The modifications include sequence insertion and deletion and other mutations in the host genomes via the error-prone non-homologous end joining （NHEJ） pathway or sequence correction or replacement through the error-free homologous recombination （HR） pathway （Symington and Gautier, 2011）. Here, we show that the CRISPR-Cas system can be applied to generate targeted gene mutations and gene corrections in plants, and the system can also be readily engi- neered to achieve deletion of large DNA fragments and for multiplex gene editing in plants.
基金Supported by the National Natural Science Foundation of China (Grant No. 30671571)BBSRC China Partnering Award (Grant No. CPA1740)
文摘The complete genome of Marek's disease virus (MDV) strain GX0101, which was integrated with the LTR sequences of REV, was cloned in Escherichia coli as a bacterial artificial chromosome (BAC). BAC vector sequences were introduced into the US2 locus of the MDV genome by homologous recombination. The viral DNA containing the BAC vector was used to transform Escherichia coli strain of DH10B. Then the recombinant virus was successfully rescued by transfection of the recombinant BAC DNA into primary chicken embryo fibroblast (CEF). This BAC viral clone was named bac-GX0101. When the reconstituted virus was inoculated into 1-day-old birds, visceral tumors could be detected as early as 62 d post infection. There was no difference in growth ability and pathogenicity to birds between the BAC derived virus and its parental virus. The BAC derived virus maintained its oncogenicity and immunosuppressive effects. In conclusion, the complete genome of GX0101 strain was successfully cloned into BAC and the infectious clone was rescued. With the powerful BAC manipulation system, the infectious clone will provide a useful tool for further understanding the functional roles of the inserted REV-LTR sequence in the GX0101 strain of MDV.
