The application of clustered regularly interspaced short palindromic repeats(CRISPR)and CRISPR-associated proteins(Cas)can be limited due to a lack of compatible protospacer adjacent motif(PAM)sequences in the DNA reg...The application of clustered regularly interspaced short palindromic repeats(CRISPR)and CRISPR-associated proteins(Cas)can be limited due to a lack of compatible protospacer adjacent motif(PAM)sequences in the DNA regions of interest.Recently,SpRY,a variant of Streptococcus pyogenes Cas9(SpCas9),was reported,which nearly completely fulfils the PAM requirement.Meanwhile,PAMs for Sp RY have not been well addressed.In our previous study,we developed the PAM Definition by Observable Sequence Excision(PAM-DOSE)and green fluorescent protein(GFP)-reporter systems to study PAMs in human cells.Herein,we endeavored to identify the PAMs of SpRY with these two methods.The results indicated that 5’-NRN-3’,5’-NTA-3’,and 5’-NCK-3’could be considered as canonical PAMs.5’-NCA-3’and 5’-NTK-3’may serve as non-priority PAMs.At the same time,PAM of 5’-NYC-3’is not recommended for human cells.These findings provide further insights into the application of SpRY for human genome editing.展开更多
Dear Editor,Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the causative agent of the global pandemic coronavirus disease 2019(COVID-19),has proven itself to be a highly virulent respiratory pathogen with...Dear Editor,Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the causative agent of the global pandemic coronavirus disease 2019(COVID-19),has proven itself to be a highly virulent respiratory pathogen with an unpredictable evolutionary capacity,posing a persistent threat to mankind.At the time of this manuscript’s publication.展开更多
基金supported by Lin HE’s Academician Workstation of New Medicine and Clinical Translation(No.18331105)the Program for Basic Science and Technology Cooperation Projects of Wenzhou City(No.H22010011),China。
文摘The application of clustered regularly interspaced short palindromic repeats(CRISPR)and CRISPR-associated proteins(Cas)can be limited due to a lack of compatible protospacer adjacent motif(PAM)sequences in the DNA regions of interest.Recently,SpRY,a variant of Streptococcus pyogenes Cas9(SpCas9),was reported,which nearly completely fulfils the PAM requirement.Meanwhile,PAMs for Sp RY have not been well addressed.In our previous study,we developed the PAM Definition by Observable Sequence Excision(PAM-DOSE)and green fluorescent protein(GFP)-reporter systems to study PAMs in human cells.Herein,we endeavored to identify the PAMs of SpRY with these two methods.The results indicated that 5’-NRN-3’,5’-NTA-3’,and 5’-NCK-3’could be considered as canonical PAMs.5’-NCA-3’and 5’-NTK-3’may serve as non-priority PAMs.At the same time,PAM of 5’-NYC-3’is not recommended for human cells.These findings provide further insights into the application of SpRY for human genome editing.
基金This work was supported by Shenzhen Science and Technology Program(JSGG20200225150431472,JSGG20210901145403012&KQTD20180411143323605)Guangdong Basic and Applied Basic Research Foundation(Grant#2020A1515110361)+3 种基金National Key Research and Development Program of China(#2021YFC0865100)Key-Area Research and Development Program of Guangdong Province(2021B1111110001)National Natural Science Foundation of China(grant#32041002,#82150206)D.G.is also supported by Guangdong Zhujiang Talents Program(#2016LJ06Y540)and National Ten-thousand Talents Program.The authors thank Lu Zhang,Ruyan Liao,Yongxia Shi and Jun Dai from Guangzhou Customs District Technology Center for the help in in vivo efficacy experiment.We also thank the other project participants,including Guanguan Li,Shuo Li and Xinjun Liu from Medi-X Pingshan,Southern University of Science and Technology for the support in preclinical study.
文摘Dear Editor,Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the causative agent of the global pandemic coronavirus disease 2019(COVID-19),has proven itself to be a highly virulent respiratory pathogen with an unpredictable evolutionary capacity,posing a persistent threat to mankind.At the time of this manuscript’s publication.
