【摘 要】
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Clustered regularly interspaced short palindromic repeats(CRISPR)-CRISPR-associated protein 9(Cas9)-mediated gene editing is revolutionizing plant research and crop breeding.Here,we present an effective and streamlined pipeline for arrayed CRISPR library
【机 构】
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National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research(Wuhan
论文部分内容阅读
Clustered regularly interspaced short palindromic repeats(CRISPR)-CRISPR-associated protein 9(Cas9)-mediated gene editing is revolutionizing plant research and crop breeding.Here,we present an effective and streamlined pipeline for arrayed CRISPR library construction and demonstrate it is suitable for small-to large-scale genome editing in plants.This pipeline introduces artificial PCR fragment-length markers for distinguishing guide RNAs(gRNAs)(FLASH),and a group of 12 constructs harboring different FLASH tags are co-transformed into plants each time.The identities of gRNAs in Agrobacterium mixtures and trans-genic plants can therefore be read out by detecting the FLASH tags,a process that requires only conven-tional PCR and gel electrophoresis rather than sequencing.We generated an arrayed CRISPR library target-ing all 1,072 members of the receptor-like kinase(RLK)family in rice.One-shot transformation generated a mutant population that covers gRNAs targeting 955 RLKs,and 74.3％(710/955)of the target genes had three or more independent T0 lines.Our results indicate that the FLASH tags act as bona fide surrogates for the gRNAs and are tightly(92.1％)associated with frameshift mutations in the target genes.In addition,the FLASH pipeline allows for rapid identification of unintended editing events without corresponding T-DNA integrations and generates high-order mutants of closely related RLK genes.Furthermore,we showed that the RLK mutant library enables rapid discovery of defense-related RLK genes.This study introduces an effective pipeline for arrayed CRISPR library construction and provides genome-wide rice RLK mutant resources for functional genomics.
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