scispace - formally typeset
Search or ask a question
Author

Guang Yang

Bio: Guang Yang is an academic researcher from ShanghaiTech University. The author has contributed to research in topics: Mutant & Genome editing. The author has an hindex of 4, co-authored 4 publications receiving 2243 citations.

Papers
More filters
Journal ArticleDOI
TL;DR: In vivo generation of mouse models carrying clinically relevant mutations using C→T and A→G editors is demonstrated, making it feasible to model and potentially cure relevant genetic diseases.
Abstract: A recently developed adenine base editor (ABE) efficiently converts A to G and is potentially useful for clinical applications. However, its precision and efficiency in vivo remains to be addressed. Here we achieve A-to-G conversion in vivo at frequencies up to 100% by microinjection of ABE mRNA together with sgRNAs. We then generate mouse models harboring clinically relevant mutations at Ar and Hoxd13, which recapitulates respective clinical defects. Furthermore, we achieve both C-to-T and A-to-G base editing by using a combination of ABE and SaBE3, thus creating mouse model harboring multiple mutations. We also demonstrate the specificity of ABE by deep sequencing and whole-genome sequencing (WGS). Taken together, ABE is highly efficient and precise in vivo, making it feasible to model and potentially cure relevant genetic diseases. CRISPR-based base editors allow for single nucleotide genome editing in a range of organisms. Here the authors demonstrate the in vivo generation of mouse models carrying clinically relevant mutations using C→T and A→G editors.

2,114 citations

Journal ArticleDOI
TL;DR: A new editing technology named as base editor for programming larger C to U (T) scope (BE-PLUS) by fusing 10 copies of GCN4 peptide to nCas9(D10A) for recruiting scFv-APOBEC-UGI-GB1 to the target sites, resulting in an increased genome-targeting scope.
Abstract: Base editor (BE), containing a cytidine deaminase and catalytically defective Cas9, has been widely used to perform base editing. However, the narrow editing window of BE limits its utility. Here, we developed a new editing technology named as base editor for programming larger C to U (T) scope (BE-PLUS) by fusing 10 copies of GCN4 peptide to nCas9(D10A) for recruiting scFv-APOBEC-UGI-GB1 to the target sites. The new system achieves base editing with a broadened window, resulting in an increased genome-targeting scope. Interestingly, the new system yielded much fewer unwanted indels and non-C-to-T conversions. We also demonstrated its potential use in gene disruption across the whole genome through induction of stop codons (iSTOP). Taken together, the BE-PLUS system offers a new editing tool with increased editing window and enhanced fidelity.

102 citations

Journal ArticleDOI
TL;DR: The initial technical assessment of applying BE3, base editor 3, in discarded human tripronuclear embryos demonstrated the BE led to highly precise and efficient genome editing in human embryos.
Abstract: CRISPR/Cas9 is a powerful tool for genome editing (Komor et al., 2017). Recently, it has been employed in several attempts to edit the human embryos (Liang et al., 2015; Kang et al., 2016; Tang et al., 2017). A major technical concern particularly relevant in studies involving human embryos is the potential off-target effects (Callaway, 2016; Plaza Reyes and Lanner, 2017). Consequently, development of safer genome editing strategy in human embryos is highly anticipated (Cyranoski and Reardon, 2015). The offtarget mutation result in part from Cas9-mediated double strand break (DSB) of DNA. Recently, base editing (BE) without the introduction of DSB has been achieved. The key design for BE is to use a catalytically inactive Cas9 to recruit the cytidine deaminase APOBEC to target sequences, leading to conversion of C to T within a window of approximately five nucleotides (Komor et al., 2016). Therefore, BE is apparently determined by additional features of the target sequence and offers a potentially safer approach for genome editing. Here we report the initial technical assessment of applying BE3, base editor 3 (Komor et al., 2016), in discarded human tripronuclear embryos. We targeted two human gene sites, HEK293 site 4 and RNF2 (Komor et al., 2016). BE3 and sgRNAs were prepared in vitro as described (Shen et al., 2014), and microinjected into the cytoplasm of the tripronuclear zygotes with the concentration of one hundred nanogram BE3 and fifty nanogram sgRNA per microliter. The zygotes were collected 48 h after microinjection, with the embryos containing different numbers of cells ranging from 1 to 8 (Table S1). In total, 8 zygotes for each of the two targets (#1–8 for HEK293 site 4, #9–16 for RNF2) were collected (Fig. 1A). Whole genome of each individual sample was amplified and used as the template for further analysis. To detect the efficiency of base editing, the region around the target sites was amplified and analyzed initially by the T7EN1 cleavage assay. For HEK293 site 4, we did not detect any cleavage bands in any of the samples (Fig. S1A). However, sequencing of the bulk PCR products revealed C to Tconversion at the sixteenth base distal from the PAM in 7 of the samples (#1–6, #8) (Figs. 1B and S1B), which is in accordance with the original report in human cell lines (Komor et al., 2016). We cloned 3 (#1–3) of the 8 bulk PCR products and sequenced multiple colonies from each primary product. For PCR products #2 and #3, each clone sequenced displayed C to T substitution, while PCR product #1 yielded one wildtype genotype besides the identical mutation genotypes (Fig. S1C), indicating highly efficient editing. To more carefully analyze the on-target editing effects, deep sequencing was applied to samples #2 and #3. In total, more than 3 M clean reads for each sample were generated. The results showed that only the 16th nucleotide distal from the PAM completely carried C to T conversion with the efficiency as high as 0.97 for sample #3, and 0.99 for sample #2. No other nucleotide alteration was detected (Fig. 1C). Besides, no on-target indel was found (Table S2). These results demonstrated the BE led to highly precise and efficient genome editing in human embryos. The same tests were performed for RNF2. T7EN1 cleavage bands were detected in 7 out of the 8 samples (#9–13, #15–16) (Fig. S2A). Sanger sequencing of PCR products confirmed C to T conversion in the 7 samples with cleavage (Figs. 1D and S2B). To further analyze the editing, 3 samples (#10–12) were selected for genotyping by TA cloning and subsequent sequencing. As reported before (Komor et al., 2016), in most cases, 2 cytosines (at the 18th and 15th nucleotide distal from the PAM) were simultaneously mutated to T, and triple C to T conversion (at the 18th, 15th, and 9th nucleotide distal from the PAM) also occurred (sample #10 and #12) (Fig. S2C). Collectively, these results demonstrated highly efficient and precise on-target base editing by BE3 in human embryos. We next tried to mutate the two genes simultaneously in the tripronuclear zygotes. To avoid possible toxicity, the concentration of each sgRNA was lowered to 25 nanogram per microliter. Nine embryos (#17–25) were collected and the target sites were analyzed by sequencing (Fig. 1A). For HEK293 site 4, the expected substitution in the sixteenth base distal from the PAM was observed in all samples (Fig. S3A), although the wild type genotype was also detectable in a few samples (Fig. S3B). A sample (#18) was randomly selected for on-target analysis by deep sequencing. The results showed that the conversion rate in the 16th C was about 0.68, which was consistent with the results of

73 citations

Journal ArticleDOI
TL;DR: The BE system induced STOP efficiently in mouse embryos and consequently in founder mice without detectable off-target, and generated isogenic single and multiplex gene mutant mice by BE-induced STOP, demonstrating the accelerated strategy in generating animal models.
Abstract: Although CRISPR/Cas9 has been widely used to generate knockout mice, two major limitations remain: the founders usually carry a mixture of genotypes, and mosaicism harboring multiple genotypes. Therefore, it takes a long time to get homozygous mutants. Recently developed base editing (BE) system, which introduces C-to-T conversion without double strand DNA cleavage, has been used to introduce artificial stop codons (i-STOP) to prematurely terminate translation, providing a cleaner strategy for genome engineering. Using this strategy, we generated CD160 KO and VISTA/CD160 double KO mice by microinjection of a single sgRNA targeting CD160 and a mixture of sgRNAs targeting VISTA and CD160, respectively. The BE system induced STOP efficiently in mouse embryos and consequently in founder mice without detectable off-target. Most interestingly, the majority of the mutants harbor same genetic modifications, indicating we generated isogenic single and multiplex gene mutant mice by BE-induced STOP. We also obtained homozygous mutant mouse in F1 mice, demonstrating the accelerated strategy in generating animal models.

10 citations


Cited by
More filters
Journal ArticleDOI
23 Nov 2017-Nature
TL;DR: Adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA are described and a transfer RNA adenosine deaminase is evolved to operate on DNA when fused to a catalytically impaired CRISPR–Cas9 mutant.
Abstract: The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.

2,451 citations

Journal ArticleDOI
TL;DR: This work analyzes key considerations when choosing genome editing agents and identifies opportunities for future improvements and applications in basic research and therapeutics.
Abstract: The development of new CRISPR-Cas genome editing tools continues to drive major advances in the life sciences. Four classes of CRISPR-Cas-derived genome editing agents-nucleases, base editors, transposases/recombinases and prime editors-are currently available for modifying genomes in experimental systems. Some of these agents have also moved rapidly into the clinic. Each tool comes with its own capabilities and limitations, and major efforts have broadened their editing capabilities, expanded their targeting scope and improved editing specificity. We analyze key considerations when choosing genome editing agents and identify opportunities for future improvements and applications in basic research and therapeutics.

1,068 citations

Journal ArticleDOI
TL;DR: A comprehensive account of the state of the art of base editing of DNA and RNA is provided, including the progressive improvements to methodologies, understanding and avoiding unintended edits, cellular and organismal delivery of editing reagents and diverse applications in research and therapeutic settings.
Abstract: RNA-guided programmable nucleases from CRISPR systems generate precise breaks in DNA or RNA at specified positions. In cells, this activity can lead to changes in DNA sequence or RNA transcript abundance. Base editing is a newer genome-editing approach that uses components from CRISPR systems together with other enzymes to directly install point mutations into cellular DNA or RNA without making double-stranded DNA breaks. DNA base editors comprise a catalytically disabled nuclease fused to a nucleobase deaminase enzyme and, in some cases, a DNA glycosylase inhibitor. RNA base editors achieve analogous changes using components that target RNA. Base editors directly convert one base or base pair into another, enabling the efficient installation of point mutations in non-dividing cells without generating excess undesired editing by-products. In this Review, we summarize base-editing strategies to generate specific and precise point mutations in genomic DNA and RNA, highlight recent developments that expand the scope, specificity, precision and in vivo delivery of base editors and discuss limitations and future directions of base editing for research and therapeutic applications.

989 citations

Journal ArticleDOI
TL;DR: The CRISPR–Cas toolkit has been expanding to include single-base editing enzymes, targeting RNA and fusing inactive Cas proteins to effectors that regulate various nuclear processes, and the new advances are considerably improving the authors' understanding of biological processes and are propelling CRISpr–Cas-based tools towards clinical use in gene and cell therapies.
Abstract: The prokaryote-derived CRISPR-Cas genome editing systems have transformed our ability to manipulate, detect, image and annotate specific DNA and RNA sequences in living cells of diverse species. The ease of use and robustness of this technology have revolutionized genome editing for research ranging from fundamental science to translational medicine. Initial successes have inspired efforts to discover new systems for targeting and manipulating nucleic acids, including those from Cas9, Cas12, Cascade and Cas13 orthologues. Genome editing by CRISPR-Cas can utilize non-homologous end joining and homology-directed repair for DNA repair, as well as single-base editing enzymes. In addition to targeting DNA, CRISPR-Cas-based RNA-targeting tools are being developed for research, medicine and diagnostics. Nuclease-inactive and RNA-targeting Cas proteins have been fused to a plethora of effector proteins to regulate gene expression, epigenetic modifications and chromatin interactions. Collectively, the new advances are considerably improving our understanding of biological processes and are propelling CRISPR-Cas-based tools towards clinical use in gene and cell therapies.

829 citations

Journal ArticleDOI
TL;DR: It is shown that expression levels are a bottleneck in base-editing efficiency, and cytidine and adenine base editors are optimized by modification of nuclear localization signals and codon usage, and ancestral reconstruction of the deaminase component.
Abstract: Base editors enable targeted single-nucleotide conversions in genomic DNA. Here we show that expression levels are a bottleneck in base-editing efficiency. We optimize cytidine (BE4) and adenine (ABE7.10) base editors by modification of nuclear localization signals (NLS) and codon usage, and ancestral reconstruction of the deaminase component. The resulting BE4max, AncBE4max, and ABEmax editors correct pathogenic SNPs with substantially increased efficiency in a variety of mammalian cell types.

572 citations