Abstract
BackgroundExpression of either Cas9 or Cas12a and guide RNAs by a single Polymerase II (Pol II) promoter represents a compact CRISPR expression system and has many advantages for different applications. In order to make this system routine in plant biology, engineering efforts are needed for developing and optimizing such single transcript unit (STU) systems for plant genome editing.ResultsTo develop novel intron-based STU (iSTU) CRISPR system (STU CRISPR 3.0), we first evaluated three introns from three plant species for carrying guide RNAs by using an enhanced green fluorescence protein (eGFP) system in rice. After validation of proper intron slicing, we inserted these gRNA-containing introns into the open reading frames (ORFs) of Cas9 and Cas12a for testing their genome editing capability. Different guide RNA processing strategies have been tested for Cas9 and Cas12a. We demonstrated singular genome editing and multiplexed genome editing with these iSTU-Cas9 and iSTU-Cas12a systems.ConclusionWe developed multiple iSTU-CRISPR/Cas9 and Cas12a systems for plant genome editing. Our results shed light on potential directions for further improvement of the iSTU systems.
Highlights
Expression of either Cas9 or Cas12a and guide RNAs by a single Polymerase II (Pol II) promoter represents a compact clustered regularly interspaced short palindromic repeats (CRISPR) expression system and has many advantages for different applications
Processing of a single guide RNA for Cas9 has been demonstrated with a hammer head (HH)-hepatitis delta virus (HDV) dual ribozyme system (Gao & Zhao. 2014), a tRNA cassette (tRNA) system (Xie et al 2015), or Csy4 which is a sequence-specific RNase (Tsai et al 2014)
Evaluation of Three Introns with an eGFP Reporter System in Rice Protoplasts To develop intron-based single transcript unit (STU) (iSTU) systems, we inserted three introns into the OFR of an enhanced green fluorescence protein gene which was drive by the maize ubiquitin 1 promoter (Fig. 1a)
Summary
Expression of either Cas or Cas12a and guide RNAs by a single Polymerase II (Pol II) promoter represents a compact CRISPR expression system and has many advantages for different applications. 2014), a tRNA system (Xie et al 2015), or Csy which is a sequence-specific RNase (Tsai et al 2014) Building on these previous studies, the first demonstration of a CRISPR/Cas STU system in plants employed HH ribozyme for precise processing of single guide RNAs (sgRNAs) (Tang et al 2016). In the case of the STU-Cas12a system, CRISPR RNA (crRNA) processing is either based on Cas12a’s self-processing of direct repeat (DR) CRISPR array (Tang et al 2019) or HH-HDV ribozyme-based processing (Wang et al 2018) In these STU systems, the gRNA cassettes were positioned at the 3′ end of the Cas gene, separated by a Poly (A) sequence or a linker sequence. Since 5′ UTR introns can often affect transcription and translation (Akua & Shaul. 2013, Cenik et al 2010, Gallegos & Rose. 2017), it adds another layer of complexity when modifying them because the compatibility of the modified 5′ UTR intron with the promoter of choice has to be empirically tested in a case by case scenario
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