Among the MB-TFs with a described function in plant development, the majority plays a role in the control of developmental stages from flower development to seed generation (SCP, bHLH155, WOUND-INDUCED POLYPEPTIDE 4, AINTEGUMENTA-LIKE 6, HRS1 HOMOLOG, NTL4, NTL8, NTL11, SHI-RELATED SEQUENCE 8, RNASE THREE-LIKE PROTEIN 2, and NF-X LIKE 2), and in root hair development or in root elongation (MAMYB, bZIP17, bZIP28, and FRF3) (Chen and McCormick, 1996 Kuusk et al., 2006 Morishita et al., 2009 Oh et al., 2010 Johansson et al., 2011 Lisso et al., 2012 Sundaram et al., 2013 Shih et al., 2014 Kim et al., 2015 Listiawan et al., 2015 Han and Krizek, 2016 Moreau et al., 2016 Tian et al., 2017 Kurt et al., 2019 Table 2). (I) mRNA accumulation for AIF, DAD1, LOX1, LOX2, LOX3, AOS, AOC1, AOC2, AOC3, AOC4, OPR3, and OPCL1 as determined by real-time quantitative (H) The AIF-C+SRDX flower that was pollinated with wild-type pollen developed elongated siliques (left), whereas short siliques (right) developed in the absence of wild-type pollen pollination. (G) The emasculated wild-type flower that was pollinated with AIF-C+SRDX pollen developed an elongated silique. (F) Close-up of a well-elongated silique (AIF-C+SRDX×AIF-C+SRDX) (left) and a short silique (right) from (E). (E) The AIF-C+SRDX flower that was manually pollinated with AIF-C+SRDX pollen developed a wellelongated silique (arrowed), whereas short siliques (s) developed without manual pollination. (D) A 50-day-old AIF-C+SRDX plant was sterile and produced short siliques. (B and C) The wild-type inflorescence (B) contained elongated siliques, whereas short and undeveloped siliques were observed in the 35S:AIF-C plant (C). (A) A 40-day-old 35S:AIF-C plant (right) was sterile and produced short siliques (arrowed), whereas a wild-type plant (left) produced a well-developed, long silique. Single plasmids containing both the gRNA and Cas protein act as all-in-one vectors, but their function is often limited to a single category (cut, nick, etc.) On the other hand, gRNA plasmids that do not co-express a Cas protein can be paired with a wide variety of Cas-containing plasmids.Phenotypic analysis of the 35S:AIF-C and AIF-C+SRDX Arabidopsis plants and the detection of gene expression in Arabidopsis plants with altered AIF expression. When using CRISPR, you will need to express both a Cas protein and a target-specific gRNA in the same cell at the same time. Select a gRNA expression plasmid based on factors such as selectable marker or cloning method. If the plasmid you’re using does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the dCas9-repressor to your specific locus. Design your gRNA to target your gene of interest’s promoter/enhancer or the beginning of the coding sequence. If the plasmid that you choose does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the dCas9-activator to your specific locus.Ĭatalytically dead dCas9, or dCas9 fused to a transcriptional repressor peptide like KRAB, can knock down gene expression by interfering with transcription. Design your gRNA sequence to direct the dCas9-activator to promoter or regulatory regions of your gene of interest. The pegRNA directs the nickase to the target site by homology to a genomic DNA locus and encodes a primer binding site and the desired edits on an RT template.Ĭatalytically dead dCas9 fused to a transcriptional activator peptide can increase transcription of a specific gene. Nickase mutants can also be used with a repair template to introduce specific edits via homology-directed repair (HDR).Ĭas9 H840A nickase fused to a reverse transcriptase (RT) is capable of installing targeted insertions, deletions, and all possible base-to-base conversions using a prime editing guide RNA (pegRNA). Double nicking strategies reduce unwanted off-target effects. These double nicks create a double-strand break (DSB) that is repaired using error-prone non-homologous end joining (NHEJ). To use a nickase mutant, you will need two gRNAs that target opposite strands of your DNA in close proximity. Adenine base editors convert adenine to inosine, which is replaced by guanosine to create A->G (or T->C on the opposite strand) mutations.ĬRISPR/Cas nickase mutants introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type Cas enzymes. Cytosine base editors convert C->T (or G->A on the opposite strand) within a small editing window specified by the gRNA. Catalytically dead dCas9 fused to a cytidine deaminase protein becomes a specific cytosine base editor that can alter DNA bases without inducing a DNA break.
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