ArticlePlant biotechnology journal2024
Guide RNA scaffold variants enabled easy cloning of large gRNA cluster for multiplexed gene editing.
Article in Plant biotechnology journal, 2024. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
What it found
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Who cites it
8 citing papers in PubMed, 15 citations in OpenAlex.
- Optimized tRNA processing and TREX2-SpCas9 fusion enable high-efficiency multiplex genome editing in plants.Plant communications · 2026Article
- Function and mechanism of double-stranded RNA-binding protein 1 in small RNA metabolism in tomato.The Plant journal : for cell and molecular biology · 2026Article
- Article
- Active Zone Maturation Controls Presynaptic Output and Release Mode and Is Regulated by Neuronal Activity.The Journal of neuroscience : the official journal of the Society for Neuroscience · 2025Article
- Turbo-charging crop improvement: harnessing multiplex editing for polygenic trait engineering and beyond.The Plant journal : for cell and molecular biology · 2025Review
- Recent advances in metabolic engineering of Escherichia coli for riboflavin biosynthesis.World journal of microbiology & biotechnology · 2025Review
- Dissecting the genetic architecture of key agronomic traits in lettuce using a MAGIC population.Genome biology · 2025Article
- Multiplex CRISPR-Cas Genome Editing: Next-Generation Microbial Strain Engineering.Journal of agricultural and food chemistry · 2024Review
Corrections and comments
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
Abstract
Cas9 protein-mediated gene editing has revolutionized genetic manipulation in most organisms. There are many cases where multiplexed gene editing is needed. Cas9 is capable of multiplex gene editing when expressed with multiple guide RNAs. Conventional cloning methods for multiplexed gene editing vector is not efficient due to repeated use of a single-guide RNA scaffold and inefficient ligation. In this study, we conducted structure-guided mutagenesis and random mutagenesis on the original sgRNA scaffold and identified a large number of functional sgRNA scaffold variants. With these scaffold variants and different tRNAs, fusion polymerase chain reaction protocol was developed to rapidly synthesize spacer-scaffold-tRNA-spacer units with up to 9 targets. In conjunction with golden gate cloning, gene editing vectors with up to 24 target sites were efficiently cloned in one-step cloning. One such gene editing vector targeting 12 genes in tomato were tested in stable transformation and 10 out of the 12 genes were found mutated in a single transgenic line. To facilitate the application of multiplexed gene editing using these scaffold variants and tRNAs from different species, a webserver was created to generate primer sets and provide template sequences for the synthesis of large sgRNA expression units based on the user-supplied target sequences and species.
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What OpenQuestion holds
Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.