Precursor-mRNAs(pre-mRNA) can be processed into one or more mature m RNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regu...Precursor-mRNAs(pre-mRNA) can be processed into one or more mature m RNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regulation, such as intronmediated enhancement(IME), whereas alternative splicing(AS) dramatically increases the protein diversity and gene functional regulation. However, the unavailability of mutants for individual spliced isoforms in plants has been a major limitation in studying the function of mRNA splicing. Here, we describe an efficient tool for manipulating the splicing of plant genes. Using a Cas9-directed base editor, we converted the 5′ splice sites in four Arabidopsis genes from the activated GT form to the inactive AT form. Silencing the AS of HAB 1.1(encoding a type 2 C phosphatase) validated its function in abscisic acid signaling, while perturbing the AS of RS31 A revealed its functional involvement in plant response to genotoxic treatment for the first time. Lastly,altering the constitutive splicing of Act2 via base editing facilitated the analysis of IME. This strategy provides an efficient tool for investigating the function and regulation of gene splicing in plants and other eukaryotes.展开更多
MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both acade...MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both academic and commercial use.Here,we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences(YTTN)in plants.Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-Lb Cas12 a system.We develop two variants,MAD7-RR and MAD7-RVR that increase the target range of MAD7,as well as an M-AFID(a MAD7-APOBEC fusion-induced deletion)system that creates predictable deletions from 50-deaminated Cs to the MAD7-cleavage site.Moreover,we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs.Using the CRISPR-MAD7 system,we have obtained regenerated mutant rice and wheat plants with up to 65.6%efficiency.展开更多
基金supported by grants from the National Key Research and Development Program of China (2016YFD0101804)the National Natural Science Foundation of China (31788103 and 31420103912)as well as the Chinese Academy of Sciences (QYZDY-SSWSMC030 and GJHZ1602)
文摘Precursor-mRNAs(pre-mRNA) can be processed into one or more mature m RNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regulation, such as intronmediated enhancement(IME), whereas alternative splicing(AS) dramatically increases the protein diversity and gene functional regulation. However, the unavailability of mutants for individual spliced isoforms in plants has been a major limitation in studying the function of mRNA splicing. Here, we describe an efficient tool for manipulating the splicing of plant genes. Using a Cas9-directed base editor, we converted the 5′ splice sites in four Arabidopsis genes from the activated GT form to the inactive AT form. Silencing the AS of HAB 1.1(encoding a type 2 C phosphatase) validated its function in abscisic acid signaling, while perturbing the AS of RS31 A revealed its functional involvement in plant response to genotoxic treatment for the first time. Lastly,altering the constitutive splicing of Act2 via base editing facilitated the analysis of IME. This strategy provides an efficient tool for investigating the function and regulation of gene splicing in plants and other eukaryotes.
基金supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences(Precision Seed Design and Breeding,XDA24020101 and XDA24020310)the National Natural Science Foundation of China(31672015,31788103)the Youth Innovation Promotion Association of the Chinese Academy of Sciences(2020000003)。
文摘MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both academic and commercial use.Here,we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences(YTTN)in plants.Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-Lb Cas12 a system.We develop two variants,MAD7-RR and MAD7-RVR that increase the target range of MAD7,as well as an M-AFID(a MAD7-APOBEC fusion-induced deletion)system that creates predictable deletions from 50-deaminated Cs to the MAD7-cleavage site.Moreover,we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs.Using the CRISPR-MAD7 system,we have obtained regenerated mutant rice and wheat plants with up to 65.6%efficiency.
