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PubMed2025

Genome editing with programmable base editors in human cells.

Genome editing has garnered significant attention over the last decade, resulting in a massive expansion of the genome engineering toolbox. Base editors encompass a class of tools that enable installing single-nucleotide changes in genomic DNA without the use of double-strand breaks. With the ever-increasing development of new and/or improved base editor systems, it is easy to be overwhelmed by the abundance of options. Here, we provide clear guidance to facilitate the selection of a base editor and to design guide RNAs (gRNAs) to suit various needs. Additionally, we describe in detail how to generate gRNA plasmids, transfect various mammalian cell types, and evaluate editing efficiencies. Finally, we give alternative methods and troubleshooting tips for some common pitfalls encountered during base editing.

Humans↗

PubMed2002

Characterization of two kinds of subgenomic RNAs produced by citrus leaf blotch virus.

Citrus leaf blotch virus (CLBV) has a single-stranded, positive-sense, genomic RNA (gRNA) organized in three ORFs, which encode a polyprotein involved in replication (RP), a potential movement protein (MP), and coat protein (CP). Northern blot hybridization of total, virion, or double-stranded RNA with probes of different gRNA regions revealed that CLBV produces two 3'-coterminal and two 5'-coterminal subgenomic RNAs (sgRNAs). The 3'-coterminal sgRNAs contain the MP (3'MP sgRNA) and CP (3'CP sgRNA) genes and untranslated regions (UTRs) of 123 and 284 nt, respectively, at their 5' end. These sgRNAs start with a hexanucleotide which is also present at the 5' terminus of the gRNA. The 5'-coterminal sgRNAs have 6795 and 5798 nt, colinear with the gRNA, and contain ORF1 and most MP gene (5'RPMP sgRNA) and most ORF1 (5'RP sgRNA), respectively. Their 3' termini map 35 and 40 nt upstream of the transcription initiation of the 3'CP and 3'MP sgRNAs, respectively, next to a potential promoter element. Our results suggest that, as in alphaviruses, CLBV internal genes are expressed via 3'-coterminal sgRNAs transcribed from the minus gRNA strand. The 5'-coterminal sgRNAs may result from early termination of the gRNA during the plus-strand synthesis.

Base Sequence↗

PubMed1992

Formation of guide RNA/messenger RNA chimeric molecules in vitro, the initial step of RNA editing, is dependent on an anchor sequence.

Synthetic pre-edited messenger RNA (mRNA) and guide RNA (gRNA) for the 5'-edited maxicircle-encoded ND7 cryptogene from Leishmania tarentolae formed chimeric molecules upon incubation in the presence of a mitochondrial extract. These chimeric molecules consisted of the gRNAs covalently linked to the mRNAs by short oligo(U) tails at normal editing sites in most cases. Unlike the previously reported chimeras present in steady-state kinetoplast RNA, the in vitro-synthesized chimeras showed no editing of downstream editing sites. The synthesis of chimeric RNAs required ATP and was dependent on the formation of a gRNA/mRNA anchor duplex 3' of the pre-edited region, as shown by in vitro mutagenesis of the mRNA and the gRNA. mRNA sequences 3' and 5' of the pre-edited region also affected the efficiency of the chimera-forming activity. This in vitro system may accurately represent the initial step in RNA editing.

Animals↗

PubMed1999

Primary and secondary structural elements required for synthesis of barley yellow dwarf virus subgenomic RNA1.

Barley yellow dwarf luteovirus (BYDV) generates three 3'-coterminal subgenomic RNAs (sgRNAs) in infected cells. The promoter of sgRNA1 is a putative hot spot for RNA recombination in luteovirus evolution. The sgRNA1 transcription start site was mapped previously to either nucleotide 2670 or nucleotide 2769 of BYDV genomic RNA (gRNA) in two independent studies. Our data support the former initiation site. The boundaries of the sgRNA1 promoter map between nucleotides 2595 and 2692 on genomic RNA. Computer prediction, phylogenetic comparison, and structural probing revealed two stem-loops (SL1 and SL2) in the sgRNA1 promoter region on the negative strand. Promoter function was analyzed by inoculating protoplasts with a full-length infectious clone of the BYDV genome containing mutations in the sgRNA promoter. Because the promoter is located in an essential coding region of the replicase gene, we duplicated it in a nonessential part of the genome from which a new sgRNA was expressed. Mutational analysis revealed that secondary structure, but not the nucleotide sequence, was important at the base of SL1. Regions with both RNA primary and secondary structural features that contributed to transcription initiation were found at the top of SL1. Primary sequence, but not the secondary structure, was required in SL2, which includes the initiation site. Disruption of base pairing near the sgRNA1 start site increased the level of transcription three- to fourfold. We propose that both primary and secondary structures of the sgRNA1 promoter of BYDV play unique roles in sgRNA1 promoter recognition and transcription initiation.

Base Sequence↗

PubMed2002

A six-nucleotide segment within the 3' untranslated region of hibiscus chlorotic ringspot virus plays an essential role in translational enhancement.

RNA plant viruses use various translational regulatory mechanisms to control their gene expression. Translational enhancement of viral mRNAs that leads to higher levels of protein synthesis from specific genes may be essential for the virus to successfully compete for cellular translational machinery. The control elements have yet to be analyzed for members of the genus Carmovirus, a small group of plant viruses with positive-sense RNA genomes. In this study, we examined the 3' untranslated region (UTR) of hibiscus chlorotic ringspot virus (HCRSV) genomic RNA (gRNA) and subgenomic RNA (sgRNA) for its role in the translational regulation of viral gene expression. The results showed that the 3' UTR of HCRSV significantly enhanced the translation of several open reading frames on gRNA and sgRNA and a viral gene in a bicistronic construct with an inserted internal ribosome entry site. Through deletion and mutagenesis studies of both the bicistronic construct and full-length gRNA, we demonstrated that a six-nucleotide sequence, GGGCAG, that is complementary to the 3' region of the 18S rRNA and a minimal length of 180 nucleotides are required for the enhancement of translation induced by the 3' UTR.

3' Untranslated Regions↗

PubMed1991

Defective RNAs of clover yellow mosaic virus encode nonstructural/coat protein fusion products.

A small group of 1.2-kb RNAs present on polyribosomes from clover yellow mosaic virus (CYMV)-infected tissue contains sequences from the genomic RNA (gRNA) of CYMV and is encapsidated by CYMV coat protein. Some features of these RNAs suggest that they are similar to defective interfering (DI) RNAs, and would be the first to be reported for the potexvirus group. The prototype 1.2-kb RNA is 1172 nucleotides in length excluding a probable poly(A) tail and is composed of two noncontiguous regions corresponding to 757 nucleotides of the 5' and 415 nucleotides of the 3' termini of CYMV's gRNA. The sequence of the prototype 1.2-kb RNA reveals that the two terminal gRNA regions present in this RNA encode a single open reading frame (ORF) joining the N-terminus of the 191-kDa nonstructural product and the C-terminus of the coat protein to form a 35-kDa 191-kDa/coat protein fusion product. The coding properties of this prototype RNA have been confirmed by translation in vitro of native and synthetic transcripts of the 1.2-kb RNAs, both of which direct the synthesis of the anticipated 35-kDa product which reacts with anti-CYMV antiserum. Three additional 1.2-kb RNA species, each of which contains a unique junction site, have been characterized. In all cases, a fusion ORF encoding a 191-kDa/coat protein fusion product is encoded on the RNA. The presence of a fusion ORF in all members of the 1.2-kb RNA species analyzed suggests that maintenance of this ORF may be important for the survival of this class of RNA within the plant. This coding strategy represents a novel property of plant virus defective RNAs.

Amino Acid Sequence↗