CRISPR/Cas12a mediated genome engineering in photosynthetic bacteria

ABSTRACTPurple non-sulfur photosynthetic bacteria (PNSB) such as R. capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. In this study, we sought to develop the class II RNA-guided CRISPR/Cas12a system from Francisella novicida for both genome editing and gene down-regulation in R. capsulatus. About 90% editing efficiency was achieved by using CRISPR/Cas12a driven by a strong promoter Ppuc when targeting ccoO or nifH gene. When both genes were simultaneously targeted, the multiplex gene editing efficiency reached >63%. In addition, CRISPR interference using deactivated Cas12a was also evaluated using reporter genes gfp and lacZ, and the repression efficiency reached >80%. In summary, our work represents the first report to develop CRISPR/Cas12a mediated genome editing/transcriptional repression in R. capsulatus, which would greatly accelerate PNSB-related researches.IMPORTANCEPurple non-sulfur photosynthetic bacteria (PNSB) such as R. capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. However, lack of efficient gene editing tools remains a main obstacle for progressing in PNSB-related researches. Here, we developed CRISPR/Cas12a for genome editing via the non-homologous end joining (NHEJ) repair machinery in R. capsulatus. In addition, DNase-deactivated Cas12a was found to simultaneously suppress multiple targeted genes. Taken together, our work offers a new set of tools for efficient genome engineering in PNSB such as R. capsulatus.

Efficient genome editing and gene knockout in Setaria viridis with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway

Plant Biotechnology ◽

10.5511/plantbiotechnology.21.0407a ◽

2021 ◽

Author(s):

Marcos Fernando Basso ◽

Karoline Estefani Duarte ◽

Thais Ribeiro Santiago ◽

Wagner Rodrigo de Souza ◽

Bruno de Oliveira Garcia ◽

...

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Gene Knockout ◽

Setaria Viridis ◽

End Joining ◽

CRISPR-Cas9: A Genome Editing Tool in Crop Plants: A Review

Agricultural Reviews ◽

10.18805/ag.r-2298 ◽

2021 ◽

Author(s):

Varsha Kumari ◽

Priyanka Kumawat ◽

Sharanabasappa Yeri ◽

Shyam Singh Rajput

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Crop Improvement ◽

End Joining ◽

Homology Directed Repair ◽

Ligase Iv ◽

A Genome ◽

Target Dna ◽

Dna Strand

Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease 9 (CRISPR-Cas9) system is a rapid technology for gene editing. CRISPR-Cas9 is an RNA guided gene editing tool where Cas9 acts as endonuclease by cutting the target DNA strand. Double Stranded Breaks (DBS) can be repaired by non-homologous end joining (NHEJ) and homology-directed repair (HDR). The NHEJ employs DNA ligase IV to rejoin the broken ends which cause insertion or deletion mutations, whereas HDR repairs the DSBs based on a homologous complementary template and results in perfect repair of broken ends. CRISPR-Cas9 impart diverse advantageous features in contrast with the conventional methods. In this review article, we have discussed CRISPR-Cas9 based genome editing along with its mechanism of action and role in crop improvement.

A genetically encoded inhibitor of 53BP1 to stimulate homology-based gene editing

10.1101/060954 ◽

2016 ◽

Cited By ~ 2

Author(s):

Marella D. Canny ◽

Leo C.K. Wan ◽

Amélie Fradet-Turcotte ◽

Alexandre Orthwein ◽

Nathalie Moatti ◽

...

Keyword(s):

Genome Editing ◽

Genome Engineering ◽

Cell Types ◽

Strand Break ◽

End Joining ◽

Dsb Repair ◽

G1 Cells ◽

Programmable Nucleases ◽

End Resection

AbstractThe expanding repertoire of programmable nucleases such as Cas9 brings new opportunities in genetic medicine1–3. In many cases, these nucleases are engineered to induce a DNA double-strand break (DSB) to stimulate precise genome editing by homologous recombination (HR). However, HR efficiency is nearly always hindered by competing DSB repair pathways such as non-homologous end-joining (NHEJ). HR is also profoundly suppressed in non-replicating cells, thus precluding the use of homology-based genome engineering in a wide variety4 of cell types. Here, we report the development of a genetically encoded inhibitor of 53BP1 (known as TP53BP1), a regulator of DSB repair pathway choice5. 53BP1 promotes NHEJ over HR by suppressing end resection, the formation of 3-prime single-stranded DNA tails, which is the rate-limiting step in HR initiation. 53BP1 also blocks the recruitment of the HR factor BRCA1 to DSB sites in G1 cells4,6. The inhibitor of 53BP1 (or i53) was identified through the screening of a massive combinatorial library of engineered ubiquitin variants by phage display7. i53 binds and occludes the ligand binding site of the 53BP1 Tudor domain with high affinity and selectivity, blocking its ability to accumulate at sites of DNA damage. i53 is a potent selective inhibitor of 53BP1 and enhances gene targeting and chromosomal gene conversion, two HR-dependent reactions. Finally, i53 can also activate HR in G1 cells when combined with the activation of end-resection and KEAP1 inhibition. We conclude that 53BP1 inhibition is a robust tool to enhance precise genome editing by canonical HR pathways.

Comparative Analysis of Genome Editors Efficiency on a Model of Mice Zygotes Microinjection

International Journal of Molecular Sciences ◽

10.3390/ijms221910221 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10221

Author(s):

Olga A. Averina ◽

Oleg A. Permyakov ◽

Olga O. Grigorieva ◽

Alexey S. Starshin ◽

Alexander M. Mazur ◽

...

Keyword(s):

Comparative Analysis ◽

Homologous Recombination ◽

Functional Genomics ◽

Genome Editing ◽

Genetic Variants ◽

Genome Engineering ◽

End Joining ◽

The Creation ◽

Indispensable Tool

Genome editing is an indispensable tool for functional genomics. The caveat of the genome-editing pipeline is a prevalence of error-prone non-homologous end joining over homologous recombination, while only the latter is suitable to introduce particularly desired genetic variants. To overcome this problem, a toolbox of genome engineering was appended by a variety of improved instruments. In this work, we compared the efficiency of a number of recently suggested improved systems for genome editing applied to the same genome regions on a murine zygote model via microinjection. As a result, we observed that homologous recombination utilizing an ssDNA template following sgRNA directed Cas9 cleavage is still the method of choice for the creation of animals with precise genome alterations.

An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

Molecular Biology Reports ◽

10.1007/s11033-020-06125-8 ◽

2021 ◽

Author(s):

Eugene V. Gasanov ◽

Justyna Jędrychowska ◽

Michal Pastor ◽

Malgorzata Wiweger ◽

Axel Methner ◽

...

Keyword(s):

Genome Editing ◽

High Efficiency ◽

Target Site ◽

Proof Of Concept ◽

Intervention Site ◽

End Joining ◽

Guide Rna ◽

Guide Rnas ◽

Cas9 Nuclease ◽

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

Use of single guided Cas9 nickase to facilitate precise and efficient genome editing in human iPSCs

Scientific Reports ◽

10.1038/s41598-021-89312-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Pan P. Li ◽

Russell L. Margolis

Keyword(s):

Genome Editing ◽

Disease Modeling ◽

Neurodegenerative Disorder ◽

Cag Repeat ◽

Spinocerebellar Ataxia Type ◽

End Joining ◽

Human Ipscs ◽

Transient Overexpression ◽

Donor Template

AbstractCas9 nucleases permit rapid and efficient generation of gene-edited cell lines. However, in typical protocols, mutations are intentionally introduced into the donor template to avoid the cleavage of donor template or re-cleavage of the successfully edited allele, compromising the fidelity of the isogenic lines generated. In addition, the double-stranded breaks (DSBs) used for editing can introduce undesirable “on-target” indels within the second allele of successfully modified cells via non-homologous end joining (NHEJ). To address these problems, we present an optimized protocol for precise genome editing in human iPSCs that employs (1) single guided Cas9 nickase to generate single-stranded breaks (SSBs), (2) transient overexpression of BCL-XL to enhance survival post electroporation, and (3) the PiggyBac transposon system for seamless removal of dual selection markers. We have used this method to modify the length of the CAG repeat contained in exon 7 of PPP2R2B. When longer than 43 triplets, this repeat causes the neurodegenerative disorder spinocerebellar ataxia type 12 (SCA12); our goal was to seamlessly introduce the SCA12 mutation into a human control iPSC line. With our protocol, ~ 15% of iPSC clones selected had the desired gene editing without “on target” indels or off-target changes, and without the deliberate introduction of mutations via the donor template. This method will allow for the precise and efficient editing of human iPSCs for disease modeling and other purposes.

Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing

Genome Biology ◽

10.1186/s13059-018-1518-x ◽

2018 ◽

Vol 19 (1) ◽

Cited By ~ 38

Author(s):

Tao Guo ◽

Yi-Li Feng ◽

Jing-Jing Xiao ◽

Qian Liu ◽

Xiu-Na Sun ◽

...

Keyword(s):

Genome Editing ◽

End Joining ◽

Genome editing for precision crop breeding - Burleigh Dodds Series in Agricultural Science ◽

Double strand break (DSB) repair pathways in plants and their application in genome engineering

10.19103/as.2020.0082.04 ◽

2021 ◽

pp. 27-62

Author(s):

Natalja Beying ◽

◽

Carla Schmidt ◽

Holger Puchta ◽

◽

...

Keyword(s):

Genome Engineering ◽

Plant Cells ◽

Strand Break ◽

End Joining ◽

Dsb Repair ◽

Strand Breaks ◽

Repair Mechanisms ◽

Repair Pathways ◽

Underlying Mechanisms

In genome engineering, after targeted induction of double strand breaks (DSBs) researchers take advantage of the organisms’ own repair mechanisms to induce different kinds of sequence changes into the genome. Therefore, understanding of the underlying mechanisms is essential. This chapter will review in detail the two main pathways of DSB repair in plant cells, non-homologous end joining (NHEJ) and homologous recombination (HR) and sum up what we have learned over the last decades about them. We summarize the different models that have been proposed and set these into relation with the molecular outcomes of different classes of DSB repair. Moreover, we describe the factors that have been identified to be involved in these pathways. Applying this knowledge of DSB repair should help us to improve the efficiency of different types of genome engineering in plants.

Advances in therapeutic application of CRISPR-Cas9

Briefings in Functional Genomics ◽

10.1093/bfgp/elz031 ◽

2019 ◽

Vol 19 (3) ◽

pp. 164-174 ◽

Cited By ~ 3

Author(s):

Jinyu Sun ◽

Jianchu Wang ◽

Donghui Zheng ◽

Xiaorong Hu

Keyword(s):

Gene Therapy ◽

Genome Editing ◽

Malignant Tumor ◽

Gene Editing ◽

Genome Engineering ◽

Therapeutic Application ◽

Adaptive Immune ◽

Efficient Gene

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is one of the most versatile and efficient gene editing technologies, which is derived from adaptive immune strategies for bacteria and archaea. With the remarkable development of programmable nuclease-based genome engineering these years, CRISPR-Cas9 system has developed quickly in recent 5 years and has been widely applied in countless areas, including genome editing, gene function investigation and gene therapy both in vitro and in vivo. In this paper, we briefly introduce the mechanisms of CRISPR-Cas9 tool in genome editing. More importantly, we review the recent therapeutic application of CRISPR-Cas9 in various diseases, including hematologic diseases, infectious diseases and malignant tumor. Finally, we discuss the current challenges and consider thoughtfully what advances are required in order to further develop the therapeutic application of CRISPR-Cas9 in the future.

CRISPR-Cas9 System for Plant Genome Editing: Current Approaches and Emerging Developments

Agronomy ◽

10.3390/agronomy10071033 ◽

2020 ◽

Vol 10 (7) ◽

pp. 1033 ◽

Cited By ~ 3

Author(s):

Jake Adolf V. Montecillo ◽

Luan Luong Chu ◽

Hanhong Bae

Keyword(s):

Genetic Engineering ◽

Genome Editing ◽

Gene Editing ◽

Fine Tuning ◽

Plant Genome ◽

Detection Methods ◽

Editing Efficiency ◽

Targeted Gene Editing ◽

Transgene Detection ◽

Selection Of

Targeted genome editing using CRISPR-Cas9 has been widely adopted as a genetic engineering tool in various biological systems. This editing technology has been in the limelight due to its simplicity and versatility compared to other previously known genome editing platforms. Several modifications of this editing system have been established for adoption in a variety of plants, as well as for its improved efficiency and portability, bringing new opportunities for the development of transgene-free improved varieties of economically important crops. This review presents an overview of CRISPR-Cas9 and its application in plant genome editing. A catalog of the current and emerging approaches for the implementation of the system in plants is also presented with details on the existing gaps and limitations. Strategies for the establishment of the CRISPR-Cas9 molecular construct such as the selection of sgRNAs, PAM compatibility, choice of promoters, vector architecture, and multiplexing approaches are emphasized. Progress in the delivery and transgene detection methods, together with optimization approaches for improved on-target efficiency are also detailed in this review. The information laid out here will provide options useful for the effective and efficient exploitation of the system for plant genome editing and will serve as a baseline for further developments of the system. Future combinations and fine-tuning of the known parameters or factors that contribute to the editing efficiency, fidelity, and portability of CRISPR-Cas9 will indeed open avenues for new technological advancements of the system for targeted gene editing in plants.