skip to main content


Title: Targeted editing and evolution of engineered ribosomes in vivo by filtered editing
Abstract

Genome editing technologies introduce targeted chromosomal modifications in organisms yet are constrained by the inability to selectively modify repetitive genetic elements. Here we describe filtered editing, a genome editing method that embeds group 1 self-splicing introns into repetitive genetic elements to construct unique genetic addresses that can be selectively modified. We introduce intron-containing ribosomes into theE. coligenome and perform targeted modifications of these ribosomes using CRISPR/Cas9 and multiplex automated genome engineering. Self-splicing of introns post-transcription yields scarless RNA molecules, generating a complex library of targeted combinatorial variants. We use filtered editing to co-evolve the 16S rRNA to tune the ribosome’s translational efficiency and the 23S rRNA to isolate antibiotic-resistant ribosome variants without interfering with native translation. This work sets the stage to engineer mutant ribosomes that polymerize abiological monomers with diverse chemistries and expands the scope of genome engineering for precise editing and evolution of repetitive DNA sequences.

 
more » « less
Award ID(s):
1740549 1935120
NSF-PAR ID:
10361558
Author(s) / Creator(s):
; ; ;
Publisher / Repository:
Nature Publishing Group
Date Published:
Journal Name:
Nature Communications
Volume:
13
Issue:
1
ISSN:
2041-1723
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Chloroplast genomes have been widely used in studying plant phylogeny and evolution. Several chloroplast genome visualization tools have been developed to display the distribution of genes on the genome. However, these tools do not draw features, such as exons, introns, repetitive elements, and variable sites, disallowing in‐depth examination of the genome structures. Here, we developed and validated a software package called Chloroplast Genome Viewers (CPGView). CPGView can draw three maps showing (i) the distributions of genes, variable sites, and repetitive sequences, including microsatellites, tandem and dispersed repeats; (ii) the structure of the cis‐splicing genes after adjusting the exon‐intron boundary positions using a coordinate scaling algorithm, and (iii) the structure of the trans‐splicing generps12. To test the accuracy of CPGView, we sequenced, assembled, and annotated 31 chloroplast genomes from 31 genera of 22 families. CPGView drew maps correctly for all the 31 chloroplast genomes. Lastly, we used CPGView to examine 5998 publicly released chloroplast genomes from 2513 genera of 553 families. CPGView succeeded in plotting maps for 5882 but failed to plot maps for 116 chloroplast genomes. Further examination showed that the annotations of these 116 genomes had various errors needing manual correction. The test on newly generated data and publicly available data demonstrated the ability of CPGView to identify errors in the annotations of chloroplast genomes. CPGView will become a widely used tool to study the detailed structure of chloroplast genomes. The web version of CPGView can be accessed fromhttp://www.1kmpg.cn/cpgview.

     
    more » « less
  2. Abstract

    CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis- and trans-DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2–18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome-editing proteins.

     
    more » « less
  3. Abstract Background

    Cas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 for genome editing in human cells, animals, and plants.

    Results

    To improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis inE. coliand identify 1977 positive point mutations of LbCas12a. We selectively assess the editing efficiency of 56 LbCas12a variants in human cells, identifying an optimal LbCas12a variant (RVQ: G146R/R182V/E795Q) with the most robust editing activity. We further test LbCas12a-RV, LbCas12a-RRV, and LbCas12a-RVQ in plants and find LbCas12a-RV has robust editing activity in rice and tomato protoplasts. Interestingly, LbCas12a-RRV, resulting from the stacking of RV and D156R, displays improved editing efficiency in stably transformed rice and poplar plants, leading to up to 100% editing efficiency inT0plants of both plant species. Moreover, this high-efficiency editing occurs even at the non-canonical TTV PAM sites.

    Conclusions

    Our results demonstrate that LbCas12a-RVQ is a powerful tool for genome editing in human cells while LbCas12a-RRV confers robust genome editing in plants. Our study reveals the tremendous potential of these LbCas12a variants for advancing precision genome editing applications across a wide range of organisms.

     
    more » « less
  4. Udall, J (Ed.)
    Abstract Availability of readily transformable germplasm, as well as efficient pipelines for gene discovery are notable bottlenecks in the application of genome editing in potato. To study and introduce traits such as resistance against biotic and abiotic factors, tuber quality traits and self-fertility, model germplasm that is amenable to gene editing and regeneration is needed. Cultivated potato is a heterozygous autotetraploid and its genetic redundancy and complexity makes studying gene function challenging. Genome editing is simpler at the diploid level, with fewer allelic variants to consider. A readily transformable diploid potato would be further complemented by genomic resources that could aid in high throughput functional analysis. The heterozygous Solanum tuberosum Group Phureja clone 1S1 has a high regeneration rate, self-fertility, desirable tuber traits and is amenable to Agrobacterium-mediated transformation. We leveraged its amenability to Agrobacterium-mediated transformation to create a Cas9 constitutively expressing line for use in viral vector-based gene editing. To create a contiguous genome assembly, a homozygous doubled monoploid of 1S1 (DM1S1) was sequenced using 44 Gbp of long reads generated from Oxford Nanopore Technologies (ONT), yielding a 736 Mb assembly that encoded 31,145 protein-coding genes. The final assembly for DM1S1 represents a nearly complete genic space, shown by the presence of 99.6% of the genes in the Benchmarking Universal Single Copy Orthologs (BUSCO) set. Variant analysis with Illumina reads from 1S1 was used to deduce its alternate haplotype. These genetic and genomic resources provide a toolkit for applications of genome editing in both basic and applied research of potato. 
    more » « less
  5. Abstract

    Base‐editing technologies enable the introduction of point mutations at targeted genomic sites in mammalian cells, with higher efficiency and precision than traditional genome‐editing methods that use DNA double‐strand breaks, such as zinc finger nucleases (ZFNs), transcription‐activator‐like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR‐associated protein 9 (CRISPR‐Cas9) system. This allows the generation of single‐nucleotide‐variant isogenic cell lines (i.e., cell lines whose genomic sequences differ from each other only at a single, edited nucleotide) in a more time‐ and resource‐effective manner. These single‐nucleotide‐variant clonal cell lines represent a powerful tool with which to assess the functional role of genetic variants in a native cellular context. Base editing can therefore facilitate genotype‐to‐phenotype studies in a controlled laboratory setting, with applications in both basic research and clinical applications. Here, we provide optimized protocols (including experimental design, methods, and analyses) to design base‐editing constructs, transfect adherent cells, quantify base‐editing efficiencies in bulk, and generate single‐nucleotide‐variant clonal cell lines. © 2020 Wiley Periodicals LLC.

    Basic Protocol 1: Design and production of plasmids for base‐editing experiments

    Basic Protocol 2: Transfection of adherent cells and harvesting of genomic DNA

    Basic Protocol 3: Genotyping of harvested cells using Sanger sequencing

    Alternate Protocol 1: Next‐generation sequencing to quantify base editing

    Basic Protocol 4: Single‐cell isolation of base‐edited cells using FACS

    Alternate Protocol 2: Single‐cell isolation of base‐edited cells using dilution plating

    Basic Protocol 5: Clonal expansion to generate isogenic cell lines and genotyping of clones

     
    more » « less