Optimizing multiplex CRISPR/Cas9-based genome editing for wheat

AbstractBackgroundCRISPR/Cas9-based genome editing holds great promise to accelerate the development of new crop varieties by providing a powerful tool to modify the genomic regions controlling major agronomic traits. To diversify the set of tools available for wheat genome engineering, we have established a tRNA-based multiplex gene editing strategy for hexaploid wheat.ResultsThe functionality of the various CRISPR/Cas9 components was assessed using the transient expression in the wheat protoplasts followed by next-generation sequencing (NGS) of the targeted genomic regions. The efficiency of wheat codon-optimized Cas9 for targeted gene editing in wheat was validated. Multiple single guide RNAs (gRNAs) were evaluated for the ability to edit the homoeologous copies of four genes affecting some important agronomic traits in wheat. Low correspondence was found between the gRNA efficiency predicted bioinformatically and that assessed in the transient expression assay. A multiplex gene editing construct with several gRNA-tRNA units under the control of a single promoter for the RNA polymerase III generated indels at the targets sites with the efficiency comparable to that obtained for a single gRNA construct.ConclusionsBy integrating the protoplast transformation assay with multiplexed NGS, it is possible to perform fast functional screens for a large number of gRNAs and to optimize constructs for effective editing of multiple independent targets in the wheat genome. The multiplexing capacity of the tandemly arrayed tRNA–gRNA construct is well suited for the simultaneous editing of the redundant gene copies in the allopolyploid genomes or genomic regions beneficially affecting multiple agronomic traits. A polycistronic gene construct that can be quickly assembled using the Golden Gate reaction along with the wheat codon optimized Cas9 will further expand the set of tools available for engineering the wheat genome.

Download Full-text

Plant-based biosensors for detecting CRISPR-mediated genome engineering

10.1101/2021.09.27.461852 ◽

2021 ◽

Author(s):

Guoliang Yuan ◽

Md Mahmudul Hassan ◽

Tao Yao ◽

Haiwei Lu ◽

Michael Melesse Vergara ◽

...

Keyword(s):

Gene Regulation ◽

Genome Editing ◽

Transient Expression ◽

Gene Editing ◽

Genome Engineering ◽

Protoplast Transformation ◽

Reliable System ◽

Base Editing ◽

Detection Systems ◽

Cas9 Nuclease

CRISPR/Cas has recently emerged as the most reliable system for genome engineering in various species. However, concerns about risks associated with CRISPR/Cas9 technology are increasing on potential unintended DNA changes that might accidentally arise from CRISPR gene editing. Developing a system that can detect and report the presence of active CRIPSR/Cas tools in biological systems is therefore very necessary. Here, we developed the real-time detection systems that can spontaneously indicate CRISPR-Cas tools for genome editing and gene regulation including CRISPR/Cas9 nuclease, base editing, prime editing and CRISPRa in plants. Using the fluorescence-based molecular biosensors, we demonstrated that the activities of CRISPR/Cas9 nuclease, base editing, prime editing and CRIPSRa can be effectively detected in transient expression via protoplast transformation and leaf infiltration (in Arabidopsis, poplar, and tobacco) and stable transformation in Arabidopsis.

Download Full-text

Recent advances in CRISPR/Cas9 and applications for wheat functional genomics and breeding

aBIOTECH ◽

10.1007/s42994-021-00042-5 ◽

2021 ◽

Author(s):

Jun Li ◽

Yan Li ◽

Ligeng Ma

Keyword(s):

Functional Genomics ◽

Genome Editing ◽

Functional Annotation ◽

Genome Engineering ◽

Agronomic Traits ◽

Wheat Breeding ◽

Breeding Programs ◽

Base Editing ◽

The World ◽

World Food Security

AbstractCommon wheat (Triticum aestivum L.) is one of the three major food crops in the world; thus, wheat breeding programs are important for world food security. Characterizing the genes that control important agronomic traits and finding new ways to alter them are necessary to improve wheat breeding. Functional genomics and breeding in polyploid wheat has been greatly accelerated by the advent of several powerful tools, especially CRISPR/Cas9 genome editing technology, which allows multiplex genome engineering. Here, we describe the development of CRISPR/Cas9, which has revolutionized the field of genome editing. In addition, we emphasize technological breakthroughs (e.g., base editing and prime editing) based on CRISPR/Cas9. We also summarize recent applications and advances in the functional annotation and breeding of wheat, and we introduce the production of CRISPR-edited DNA-free wheat. Combined with other achievements, CRISPR and CRISPR-based genome editing will speed progress in wheat biology and promote sustainable agriculture.

Download Full-text

Human Genetic Diversity Alters Therapeutic Gene Editing Off-Target Outcomes

Blood ◽

10.1182/blood-2021-152429 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3993-3993

Author(s):

Linda Yingqi Lin ◽

Samuele Cancellieri ◽

Jing Zeng ◽

Francesco Masillo ◽

My Anh Nguyen ◽

...

Keyword(s):

Genetic Diversity ◽

Clinical Trials ◽

Genome Editing ◽

Gene Editing ◽

Conflicts Of Interest ◽

African Ancestry ◽

Great Promise ◽

Personal Genome ◽

Hematopoietic Stem ◽

The Impact

Abstract CRISPR gene editing holds great promise to modify somatic genomes to ameliorate disease. In silico prediction of homologous sites coupled with biochemical evaluation of possible genomic off-targets may predict genotoxicity risk of individual gene editing reagents. However, standard computational and biochemical methods focus on reference genomes and do not consider the impact of genetic diversity on off-target potential. Here we developed a web application called CRISPRme that explicitly and efficiently integrates human genetic variant datasets with orthogonal genomic annotations to predict and prioritize off-target sites at scale. The method considers both single-nucleotide variants (SNVs) and indels, accounts for bona fide haplotypes, accepts spacer:protospacer mismatches and bulges, and is suitable for personal genome analyses. We tested the tool with a guide RNA (gRNA) targeting the BCL11A erythroid enhancer that has shown therapeutic promise in clinical trials for sickle cell disease (SCD) and β-thalassemia (Frangoul et al. NEJM 2021). We find that the top predicted off-target site is produced by a non-reference allele common in African-ancestry populations (rs114518452, minor allele frequency (MAF) = 4.5%) that introduces a protospacer adjacent motif (PAM) for SpCas9. We validate that SpCas9 generates indels (~9.6% frequency) and chr2 pericentric inversions in a strictly allele-specific manner in edited CD34+ hematopoietic stem/progenitor cells (HSPCs), although a high-fidelity Cas9 variant mitigates this off-target. This report illustrates how population and private genetic variants should be considered as modifiers of genome editing outcomes. We expect that variant-aware off-target assessment will be required for therapeutic genome editing efforts going forward, including both ongoing and future clinical trials, and we provide a powerful approach for comprehensive off-target prediction. CRISPRme is available at crisprme.di.univr.it. Disclosures No relevant conflicts of interest to declare.

Download Full-text

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.

Download Full-text

MultiGuideScan: a multi-processing tool for designing CRISPR guide RNA libraries

Bioinformatics ◽

10.1093/bioinformatics/btz616 ◽

2019 ◽

Cited By ~ 1

Author(s):

Tao Li ◽

Shaokai Wang ◽

Feng Luo ◽

Fang-Xiang Wu ◽

Jianxin Wang

Keyword(s):

Gene Editing ◽

Genome Engineering ◽

Supplementary Information ◽

Guide Rna ◽

Multiple Processes ◽

Design Guide ◽

Computationally Expensive ◽

Genomic Regions ◽

Process Mode ◽

Large Genomes

Abstract Summary The recent advance in genome engineering technologies based on CRISPR/Cas9 system is enabling people to systematically understand genomic functions. A short RNA string (the CRISPR guide RNA) can guide the Cas9 endonuclease to specific locations in complex genomes to cut DNA double-strands. The CRISPR guide RNA is essential for gene editing systems. Recently, the GuideScan software is developed to design CRISPR guide RNA libraries, which can be used for genome editing of coding and non-coding genomic regions effectively. However, GuideScan is a serial program and computationally expensive for designing CRISPR guide RNA libraries from large genomes. Here, we present an efficient guide RNA library designing tool (MultiGuideScan) by implementing multiple processes of GuideScan. MultiGuideScan speeds up the guide RNA library designing about 9–12 times on a 32-process mode comparing to GuideScan. MultiGuideScan makes it possible to design guide RNA libraries from large genomes. Availability and implementation: MultiGuideScan is available at GitHub https://github.com/bioinfomaticsCSU/MultiGuideScan. Supplementary information Supplementary data are available at Bioinformatics online.

Download Full-text

CRISPR/Cas12a mediated genome engineering in photosynthetic bacteria

10.1101/2020.10.05.327569 ◽

2020 ◽

Author(s):

Yang Zhang ◽

Jifeng Yuan

Keyword(s):

Genome Editing ◽

Transcriptional Repression ◽

Gene Editing ◽

Photosynthetic Bacteria ◽

Genome Engineering ◽

Biotechnological Applications ◽

End Joining ◽

Editing Efficiency ◽

Francisella Novicida ◽

Non Homologous End Joining

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.

Download Full-text

Plant viral vectors: Expanding the Possibilities of Precise Gene Editing in Plant Genomes

10.21203/rs.3.rs-265848/v1 ◽

2021 ◽

Author(s):

Stuti Kujur ◽

Muthappa Senthil-Kumar ◽

Rahul Kumar

Keyword(s):

Genome Editing ◽

Plant Transformation ◽

Viral Vectors ◽

Gene Editing ◽

Genome Engineering ◽

Plant Cells ◽

In Planta ◽

Plant Genomes ◽

Efficient Gene ◽

Transformation Methods

Abstract The lack of a highly efficient method for delivering reagents for genome engineering to plant cells remains a bottleneck in achieving efficient gene-editing in plant genomes. A suite of recent reports uncovers the newly emerged roles of viral vectors, which can introduce gene-edits in plants with high mutation frequencies through in planta delivery. Here, we focus on the emerging protocols that utilized different approaches for virus-mediated genome editing in model plants. Testing of these protocols and the newly identified hypercompact Casɸ systems is needed to broaden the scope of genome-editing in most plant species, including crops, with minimized reliance on conventional plant transformation methods in the future.

Download Full-text

Genome Editing with CRISPR

Ethics & Medics ◽

10.5840/em20164134 ◽

2016 ◽

Vol 41 (3) ◽

pp. 1-3

Author(s):

Nicanor Pier Giorgio Austriaco ◽

Keyword(s):

Genetic Engineering ◽

Genome Editing ◽

Gene Editing ◽

Life Sciences ◽

Great Promise ◽

Bacterial Cells ◽

Kitchen Table ◽

A Genome ◽

Do It Yourself

There has been much discussion regarding the proper use of the powerful CRISPR technologies that can be used to edit the genome. CRISPR is a technique borrowed from bacterial cells that will allow scientists to quickly and precisely change the DNA of nearly any organism, including humans. Unlike other gene-editing technologies, CRISPR is cheap, quick, and easy to use. In fact, do-it-yourself CRISPR genome editing kits are available online for less than $200, which will enable anyone, including so-called biohackers, to do genetic engineering at the kitchen table. In only three years—CRISPR as a genome-editing tool was first described in 2012—it is already universally acknowledged that this technology will revolutionize the life sciences. But CRISPR’s great promise has also sparked a great ethical and societal debate on its legitimate uses, most significantly on whether it should be used to alter the genomes of our children and grandchildren.

Download Full-text

CRISPR: a journey of gene-editing based medicine

Genes & Genomics ◽

10.1007/s13258-020-01002-x ◽

2020 ◽

Vol 42 (12) ◽

pp. 1369-1380

Author(s):

Zhabiz Golkar

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Genome Engineering ◽

High Strength ◽

Evidence Based ◽

Modern Biology ◽

Scientific Communities ◽

Short Palindromic Repeat ◽

Practical Tool ◽

Based Medicine

AbstractCRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) is one of the hallmark of biological tools, contemplated as a valid and hopeful alternatives to genome editing. Advancements in CRISPR-based technologies have empowered scientists with an editing kit that allows them to employ their knowledge for deleting, replacing and lately “Gene Surgery”, and provides unique control over genes in broad range of species, and presumably in humans. These fast-growing technologies have high strength and flexibility and are becoming an adaptable tool with implementations that are altering organism’s genome and easily used for chromatin manipulation. In addition to the popularity of CRISPR in genome engineering and modern biology, this major tool authorizes breakthrough discoveries and methodological advancements in science. As scientists are developing new types of experiments, some of the applications are raising questions about what CRISPR can enable. The results of evidence-based research strongly suggest that CRISPR is becoming a practical tool for genome-engineering and to create genetically modified eukaryotes, which is needed to establish guidelines on new regulatory concerns for scientific communities.

Download Full-text

Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA

Nature Communications ◽

10.1038/ncomms12617 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 306

Author(s):

Yi Zhang ◽

Zhen Liang ◽

Yuan Zong ◽

Yanpeng Wang ◽

Jinxing Liu ◽

...

Keyword(s):

Durum Wheat ◽

Genome Editing ◽

Bread Wheat ◽

Transient Expression ◽

Plant Species ◽

Genome Engineering ◽

Plant Genome ◽

Engineering Research ◽

Plant Genomes ◽

Highly Efficient

Abstract Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research.

Download Full-text