scholarly journals Spelling Changes and Fluorescent Tagging With Prime Editing Vectors for Plants

2021 ◽  
Vol 3 ◽  
Author(s):  
Li Wang ◽  
Hilal Betul Kaya ◽  
Ning Zhang ◽  
Rhitu Rai ◽  
Matthew R. Willmann ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Binding Site ◽  
Nicotiana Benthamiana ◽  
Primer Binding Site ◽  
First Report ◽  
Guide Rna ◽  
Insertions And Deletions ◽  
Target Dna ◽  
Fluorescent Tagging

Prime editing is an adaptation of the CRISPR-Cas system that uses a Cas9(H840A)-reverse transcriptase fusion and a guide RNA amended with template and primer binding site sequences to achieve RNA-templated conversion of the target DNA, allowing specified substitutions, insertions, and deletions. In the first report of prime editing in plants, a variety of edits in rice and wheat were described, including insertions up to 15 bp. Several studies in rice quickly followed, but none reported a larger insertion. Here, we report easy-to-use vectors for prime editing in dicots as well as monocots, their validation in Nicotiana benthamiana, rice, and Arabidopsis, and an insertion of 66 bp that enabled split-GFP fluorescent tagging.

scholarly journals Spelling changes and fluorescent tagging with prime editing vectors for plants

2020 ◽  
Author(s):  
Li Wang ◽  
Hilal Betul Kaya ◽  
Ning Zhang ◽  
Rhitu Rai ◽  
Matthew R. Willmann ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Binding Site ◽  
Nicotiana Benthamiana ◽  
Primer Binding Site ◽  
First Report ◽  
Guide Rna ◽  
Insertions And Deletions ◽  
Target Dna ◽  
Fluorescent Tagging

AbstractPrime editing (PE) is a recent adaptation of the CRISPR-Cas system that uses a Cas9(H840A)-reverse transcriptase (RT) fusion and a guide RNA (pegRNA) amended with template and primer binding site (PBS) sequences to achieve RNA-templated conversion of the target DNA, allowing specified substitutions, insertions, and deletions. In the first report of PE in plants, a variety of edits in rice and wheat were described, including insertions up to 15 bp. Several studies in rice quickly followed, but none reported a larger insertion. Here, we report easy-to-use vectors for PE in dicots and monocots, their validation in Nicotiana benthamiana, rice and Arabidopsis, and an insertion of 66 bp that enabled split-GFP fluorescent tagging.

scholarly journals HIV-1 Employs Multiple Mechanisms To Resist Cas9/Single Guide RNA Targeting the Viral Primer Binding Site

2018 ◽  
Vol 92 (20) ◽  
Author(s):  
Zhen Wang ◽  
Wenzhou Wang ◽  
Ya Cheng Cui ◽  
Qinghua Pan ◽  
Weijun Zhu ◽  
...  
Keyword(s):  
Binding Site ◽  
Reverse Transcription ◽  
Gene Editing ◽  
Viral Escape ◽  
Primer Binding Site ◽  
Minus Strand ◽  
Guide Rna ◽  
Target Dna ◽  
Almost All ◽  
Hiv 1

ABSTRACTThe clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (Cas9) gene-editing technology has been used to inactivate viral DNA as a new strategy to eliminate chronic viral infections, including HIV-1. This utility of CRISPR-Cas9 is challenged by the high heterogeneity of HIV-1 sequences, which requires the design of the single guide RNA (sgRNA; utilized by the CRISPR-Cas9 system to recognize the target DNA) to match a specific HIV-1 strain in an HIV patient. One solution to this challenge is to target the viral primer binding site (PBS), which HIV-1 copies from cellular tRNA3Lysin each round of reverse transcription and is thus conserved in almost all HIV-1 strains. In this study, we demonstrate that PBS-targeting sgRNA directs Cas9 to cleave the PBS DNA, which evokes deletions or insertions (indels) and strongly diminishes the production of infectious HIV-1. While HIV-1 escapes from PBS-targeting Cas9/sgRNA, unique resistance mechanisms are observed that are dependent on whether the plus or the minus strand of the PBS DNA is bound by sgRNA. Characterization of these viral escape mechanisms will inform future engineering of Cas9 variants that can more potently and persistently inhibit HIV-1 infection.IMPORTANCEThe results of this study demonstrate that the gene-editing complex Cas9/sgRNA can be programmed to target and cleave HIV-1 PBS DNA, and thus, inhibit HIV-1 infection. Given that almost all HIV-1 strains have the same PBS, which is copied from the cellular tRNA3Lysduring reverse transcription, PBS-targeting sgRNA can be used to inactivate HIV-1 DNA of different strains. We also discovered that HIV-1 uses different mechanisms to resist Cas9/sgRNAs, depending on whether they target the plus or the minus strand of PBS DNA. These findings allow us to predict that a Cas9 variant that uses the CCA sequence as the protospacer adjacent motif (PAM) should more strongly and persistently suppress HIV-1 replication. Together, these data have identified the PBS as the target DNA of Cas9/sgRNA and have predicted how to improve Cas9/sgRNA to achieve more efficient and sustainable suppression of HIV-1 infection, therefore improving the capacity of Cas9/sgRNA in curing HIV-1 infection.

scholarly journals Mutations in the U5 Region Adjacent to the Primer Binding Site Affect tRNA Cleavage by Human Immunodeficiency Virus Type 1 Reverse Transcriptase In Vivo

2007 ◽  
Vol 82 (2) ◽  
pp. 719-727 ◽  
Author(s):  
Jangsuk Oh ◽  
Mary Jane McWilliams ◽  
John G. Julias ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Binding Site ◽  
Rnase H ◽  
Primer Binding Site ◽  
Cleavage Specificity ◽  
Immunodeficiency Virus ◽  
Trna Primer ◽  
Hiv 1

ABSTRACT In retroviruses, the first nucleotide added to the tRNA primer defines the end of the U5 region in the right long terminal repeat, and the subsequent removal of this tRNA primer by RNase H exactly defines the U5 end of the linear double-stranded DNA. In most retroviruses, the entire tRNA is removed by RNase H cleavage at the RNA/DNA junction. However, the RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase cleaves the tRNA 1 nucleotide from the RNA/DNA junction at the U5/primer binding site (PBS) junction, which leaves an rA residue at the U5 terminus. We made sequence changes at the end of the U5 region adjacent to the PBS in HIV-1 to determine whether such changes affect the specificity of tRNA primer cleavage by RNase H. In some of the mutants, RNase H usually removed the entire tRNA, showing that the cleavage specificity was shifted by 1 nucleotide. This result suggests that the tRNA cleavage specificity of the HIV-1 RNase domain H depends on sequences in U5.

scholarly journals Human immunodeficiency virus type 1 reverse transcriptase Affinity labeling of the primer binding site

FEBS Letters ◽  
1992 ◽  
Vol 312 (2-3) ◽  
pp. 249-251 ◽  
Author(s):  
R.L. Mitina ◽  
S.V. Doronin ◽  
M.I. Dobrikov ◽  
D.R. Tabatadze ◽  
A.S. Levina ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Binding Site ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Primer Binding Site ◽  
Affinity Labeling ◽  
Immunodeficiency Virus

scholarly journals A Nucleotide Substitution in the tRNALys Primer Binding Site Dramatically Increases Replication of Recombinant Simian Immunodeficiency Virus Containing a Human Immunodeficiency Virus Type 1 Reverse Transcriptase

2002 ◽  
Vol 76 (11) ◽  
pp. 5803-5806 ◽  
Author(s):  
Kelly Soderberg ◽  
Lynn Denekamp ◽  
Sarah Nikiforow ◽  
Karen Sautter ◽  
Ronald C. Desrosiers ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Binding Site ◽  
Simian Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Nucleotide Substitution ◽  
Primer Binding Site ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT A recombinant simian immunodeficiency virus (SIV) derived from strain 239 (SIVmac239) with reverse transcriptase (RT) sequences from human immunodeficiency virus type 1 (HIV-1) strain HXB2 was severely impaired for replication. Detectable p27Gag levels were not observed until day 65 and peak p27Gag levels were not reached until day 75 after transfection of CEMx174 cells with the recombinant DNA. Sequences from the latter time point did not contain amino acid substitutions in HIV-1 RT; however, a single nucleotide substitution (thymine to cytosine) was found at position eight of the SIV primer binding site. We engineered an RT/SHIV genome with the thymine-to-cytosine substitution, called RT/SHIV/TC, and observed dramatically faster replication kinetics than were observed with the parental RT/SHIV from which this variant was derived. RT/SHIV/TC provides an improved system for study of the impact of drug resistance mutations in HIV-1 RT in a relevant animal model.

scholarly journals pegFinder: A pegRNA designer for CRISPR prime editing

2020 ◽  
Author(s):  
Ryan D. Chow ◽  
Jennifer S. Chen ◽  
Johanna Shen ◽  
Sidi Chen
Keyword(s):  
Web Application ◽  
Double Strand Breaks ◽  
Primer Binding Site ◽  
Web Tool ◽  
Strand Breaks ◽  
Guide Rna ◽  
Guide Rnas ◽  
Target Dna ◽  
Target Sites ◽  
User Friendly

To the EditorCRISPR technologies have been widely adopted as powerful tools for targeted genomic manipulation 1. Recently, a new CRISPR-based strategy for precision genome editing was developed that enables diverse genomic alterations to be directly written into target sites without requiring double-strand breaks (DSBs) or donor templates 2. Termed prime editing, this approach involves two key components: 1) a catalytically impaired Cas9 nickase fused to a reverse transcriptase (PE2), and 2) a multifunctional prime editing guide RNA (pegRNA) that specifies the target site and further acts as a template for reverse transcription (RT). pegRNAs are similar to standard single-guide RNAs (sgRNAs), but additionally have a customizable extension on the 3’ end. The 3’ extension is composed of a RT template that encodes the desired edit and a primer binding site (PBS) that anneals to the target genomic site to prime the RT reaction 2. These additional components considerably increase the complexity of pegRNA design compared to standard sgRNAs. While many tools have been developed for identifying candidate sgRNAs in a target DNA sequence 3–8, no user-friendly web application currently exists for designing pegRNAs. We therefore developed pegFinder, a streamlined web tool that rapidly designs candidate pegRNAs (Figure 1). The pegFinder web portal is freely available at http://pegfinder.sidichenlab.org/ (Supplementary Figure 1).

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