A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing.
Grünewald Julian,Zhou Ronghao,Lareau Caleb A,Garcia Sara P,Iyer Sowmya,Miller Bret R,Langner Lukas M,Hsu Jonathan Y,Aryee Martin J,Joung J Keith
Nature biotechnology
Existing adenine and cytosine base editors induce only a single type of modification, limiting the range of DNA alterations that can be created. Here we describe a CRISPR-Cas9-based synchronous programmable adenine and cytosine editor (SPACE) that can concurrently introduce A-to-G and C-to-T substitutions with minimal RNA off-target edits. SPACE expands the range of possible DNA sequence alterations, broadening the research applications of CRISPR base editors.
10.1038/s41587-020-0535-y
Highly efficient single base editing in Aspergillus niger with CRISPR/Cas9 cytidine deaminase fusion.
Huang Lianggang,Dong Hongzhi,Zheng Junwei,Wang Bin,Pan Li
Microbiological research
Classic genome editing tools including ZFN, TALEN, and CRISPR/Cas9 rely on DNA double-strand breaks for genome editing. To prevent the potential hazard caused by double-strand breaks (DSBs), a series of single base editing tools that convert cytidine (C) to thymine (T) without DSBs have been developed extensively in multiple species. Herein, we report for the first time that C was converted to T with a high frequency in the filamentous fungi Aspergillus niger by fusing cytidine deaminase and Cas9 nickase. Using the CRISPR/Cas9-dependent base editor and inducing nonsense mutations via single base editing, we inactivated the uridine auxotroph gene pyrG and the pigment gene fwnA with an efficiency of 47.36%-100% in A.niger. At the same time, the single-base editing results of the non-phenotypic gene prtT showed an efficiency of 60%. The editable window reached 8 bases (from C2 to C9 in the protospacer) in A. niger. Overall, we successfully constructed a single base editing system in A. niger. This system provides a more convenient tool for investigating gene function in A. niger, and provides a new tool for genetic modification in filamentous fungi.
10.1016/j.micres.2019.03.007
The CRISPR tool kit for genome editing and beyond.
Nature communications
CRISPR is becoming an indispensable tool in biological research. Once known as the bacterial immune system against invading viruses, the programmable capacity of the Cas9 enzyme is now revolutionizing diverse fields of medical research, biotechnology, and agriculture. CRISPR-Cas9 is no longer just a gene-editing tool; the application areas of catalytically impaired inactive Cas9, including gene regulation, epigenetic editing, chromatin engineering, and imaging, now exceed the gene-editing functionality of WT Cas9. Here, we will present a brief history of gene-editing tools and describe the wide range of CRISPR-based genome-targeting tools. We will conclude with future directions and the broader impact of CRISPR technologies.
10.1038/s41467-018-04252-2
Highly efficient base editing in bacteria using a Cas9-cytidine deaminase fusion.
Zheng Ke,Wang Yang,Li Na,Jiang Fang-Fang,Wu Chang-Xian,Liu Fang,Chen Huan-Chun,Liu Zheng-Fei
Communications biology
The ability to precisely edit individual bases of bacterial genomes would accelerate the investigation of the function of genes. Here we utilized a nickase Cas9-cytidine deaminase fusion protein to direct the conversion of cytosine to thymine within prokaryotic cells, resulting in high mutagenesis frequencies in and . Our study suggests that CRISPR/Cas9-guided base-editing is a viable alternative approach to generate mutant bacterial strains.
10.1038/s42003-018-0035-5
Expanding C-T base editing toolkit with diversified cytidine deaminases.
Cheng Tian-Lin,Li Shuo,Yuan Bo,Wang Xiaolin,Zhou Wenhao,Qiu Zilong
Nature communications
Base editing tools for cytosine to thymine (C-T) conversion enable genome manipulation at single base-pair resolution with high efficiency. Available base editors (BEs) for C-T conversion (CBEs) have restricted editing scopes and nonnegligible off-target effects, which limit their applications. Here, by screening diversified lamprey cytidine deaminases, we establish various CBEs with expanded and diversified editing scopes, which could be further refined by various fusing strategies, fusing at either N-terminus or C-terminus of nCas9. Furthermore, off-target analysis reveals that several CBEs display improved fidelity. Our study expands the toolkits for C-T conversion, serves as guidance for appropriate choice and offers a framework for benchmarking future improvement of base editing tools.
10.1038/s41467-019-11562-6
CRISPR Toolbox for Genome Editing in .
Yamashita Kensuke,Iriki Hoshie,Kamimura Yoichiro,Muramoto Tetsuya
Frontiers in cell and developmental biology
The development of new techniques to create gene knockouts and knock-ins is essential for successful investigation of gene functions and elucidation of the causes of diseases and their associated fundamental cellular processes. In the biomedical model organism , the methodology for gene targeting with homologous recombination to generate mutants is well-established. Recently, we have applied CRISPR/Cas9-mediated approaches in , allowing the rapid generation of mutants by transiently expressing sgRNA and Cas9 using an all-in-one vector. CRISPR/Cas9 techniques not only provide an alternative to homologous recombination-based gene knockouts but also enable the creation of mutants that were technically unfeasible previously. Herein, we provide a detailed protocol for the CRISPR/Cas9-based method in . We also describe new tools, including double knockouts using a single CRISPR vector, drug-inducible knockouts, and gene knockdown using CRISPR interference (CRISPRi). We demonstrate the use of these tools for some candidate genes. Our data indicate that more suitable mutants can be rapidly generated using CRISPR/Cas9-based techniques to study gene function in .
10.3389/fcell.2021.721630
Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions.
Kim Y Bill,Komor Alexis C,Levy Jonathan M,Packer Michael S,Zhao Kevin T,Liu David R
Nature biotechnology
Base editing induces single-nucleotide changes in the DNA of living cells using a fusion protein containing a catalytically defective Streptococcus pyogenes Cas9, a cytidine deaminase, and an inhibitor of base excision repair. This genome editing approach has the advantage that it does not require formation of double-stranded DNA breaks or provision of a donor DNA template. Here we report the development of five C to T (or G to A) base editors that use natural and engineered Cas9 variants with different protospacer-adjacent motif (PAM) specificities to expand the number of sites that can be targeted by base editing 2.5-fold. Additionally, we engineered base editors containing mutated cytidine deaminase domains that narrow the width of the editing window from ∼5 nucleotides to as little as 1-2 nucleotides. We thereby enabled discrimination of neighboring C nucleotides, which would otherwise be edited with similar efficiency, and doubled the number of disease-associated target Cs able to be corrected preferentially over nearby non-target Cs.
10.1038/nbt.3803
Highly efficient base editing in using an engineered CRISPR RNA-guided cytidine deaminase.
Gu Tongnian,Zhao Siqi,Pi Yishuang,Chen Weizhong,Chen Chuanyuan,Liu Qian,Li Min,Han Dali,Ji Quanjiang
Chemical science
Novel therapeutic means against infections are urgently needed due to the emergence of drug-resistant . We report the development of a CRISPR RNA-guided cytidine deaminase (pnCasSA-BEC), enabling highly efficient gene inactivation and point mutations in . We engineered a fusion of a Cas9 nickase (Cas9D10A) and a cytidine deaminase (APOBEC1) that can be guided to a target genomic locus for gene inactivation generating a premature stop codon. The pnCasSA-BEC system nicks the non-edited strand of the genomic DNA, directly catalyzes the conversion of cytidine (C) to uridine (U), and relies on DNA replication to achieve C → T (G → A) conversion without using donor repair templates. The development of the base-editing system will dramatically accelerate drug-target exploration in and provides critical insights into the development of base-editing tools in other microbes.
10.1039/c8sc00637g
Programmable adenine deamination in bacteria using a Cas9-adenine-deaminase fusion.
Chemical science
Precise genetic manipulation is vital to studying bacterial physiology, but is difficult to achieve in some bacterial species due to the weak intrinsic homologous recombination (HR) capacity and lack of a compatible exogenous HR system. Here we report the establishment of a rapid and efficient method for directly converting adenine to guanine in bacterial genomes using the fusion of an adenine deaminase and a Cas9 nickase. The method achieves the conversion of adenine to guanine an enzymatic deamination reaction and a subsequent DNA replication process rather than HR, which is utilized in conventional bacterial genetic manipulation methods, thereby substantially simplifying the genome editing process. A systematic screening targeting the possibly editable adenine sites of , the importer of the staphylopine/metal complex in , pinpoints key residues for metal importation, demonstrating that application of the system would greatly facilitate the genomic engineering of bacteria.
10.1039/c9sc03784e