Cas12a mediates efficient and precise endogenous gene tagging via MITI: microhomology-dependent targeted integrations

AbstractEfficient exogenous DNA integration can be mediated by Cas9 through the non-homology end-joining pathway. However, such integrations are often imprecise and contain a variety of mutations at the junctions between the external DNA and the genomic loci. Here we describe a microhomology-dependent targeted integration method, designated MITI, for precise site-specific gene insertions. We found that the MITI strategy yielded higher knock-in accuracy than Cas9 HITI for the insertion of external DNA and tagging endogenous genes. Furthermore, in combination with negative selection and four different CrRNAs targeting donor vectors and genome-targeted sites with a CrRNA array, MITI facilitated precise ligation at all junctions. Therefore, our Cas12a-based MITI method increases the repertoire of precision genome engineering approaches and provides a useful tool for various gene editing applications.

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Efficient Gene Editing Through an Intronic Selection Marker in Cells

10.21203/rs.3.rs-739001/v1 ◽

2021 ◽

Author(s):

Shang Wang ◽

Yuqing Li ◽

Li Zhong ◽

Kai Wu ◽

Ruhua Zhang ◽

...

Keyword(s):

Gene Editing ◽

Selection Marker ◽

End Joining ◽

Dna Variation ◽

Editing Efficiency ◽

Endogenous Gene ◽

Positive Effects ◽

Efficient Gene ◽

Almost All ◽

In Cells

Abstract Background Gene editing technology has provided researchers with the ability to modify genome sequences in almost all eukaryotes. Gene-edited cell lines are being used with increasing frequency in both bench research and targeted therapy. Despite the great importance and universality of gene editing, however, precision and efficiency are hard to achieve with the prevailing editing strategies, such as homology-directed DNA repair (HDR) and the use of base editors (BEs). Results & Discussion Our group has developed a novel gene editing technology to indicate DNA variation with an independent selection marker using an HDR strategy, which we named Gene Editing through an Intronic Selection marker (GEIS). GEIS uses a simple process to avoid nonhomologous end joining (NHEJ)-mediated false-positive effects and achieves editing efficiency as high as 91% without disturbing endogenous gene splicing and expression. We re-examined the correlation of the conversion tract and editing efficiency, and our data suggest that GEIS has the potential to edit approximately 99% of gene editing targets in human and mouse cells. The results of further comprehensive analysis suggest that the strategy may be useful for introducing multiple DNA variations in cells.

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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.

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Global and Local Manipulation of DNA Repair Mechanisms to Alter Site-Specific Gene Editing Outcomes in Hematopoietic Stem Cells

Frontiers in Genome Editing ◽

10.3389/fgeed.2020.601541 ◽

2020 ◽

Vol 2 ◽

Author(s):

Elizabeth K. Benitez ◽

Anastasia Lomova Kaufman ◽

Lilibeth Cervantes ◽

Danielle N. Clark ◽

Paul G. Ayoub ◽

...

Keyword(s):

Dna Repair ◽

Gene Editing ◽

Ex Vivo ◽

Specific Gene ◽

Gene Correction ◽

End Joining ◽

Therapeutic Success ◽

Hematopoietic Stem ◽

Dna Repair Mechanisms ◽

Repair Mechanisms

Monogenic disorders of the blood system have the potential to be treated by autologous stem cell transplantation of ex vivo genetically modified hematopoietic stem and progenitor cells (HSPCs). The sgRNA/Cas9 system allows for precise modification of the genome at single nucleotide resolution. However, the system is reliant on endogenous cellular DNA repair mechanisms to mend a Cas9-induced double stranded break (DSB), either by the non-homologous end joining (NHEJ) pathway or by the cell-cycle regulated homology-directed repair (HDR) pathway. Here, we describe a panel of ectopically expressed DNA repair factors and Cas9 variants assessed for their ability to promote gene correction by HDR or inhibit gene disruption by NHEJ at the HBB locus. Although transient global overexpression of DNA repair factors did not improve the frequency of gene correction in primary HSPCs, localization of factors to the DSB by fusion to the Cas9 protein did alter repair outcomes toward microhomology-mediated end joining (MMEJ) repair, an HDR event. This strategy may be useful when predictable gene editing outcomes are imperative for therapeutic success.

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Homology arms of targeting vectors for gene insertions and CRISPR/Cas9 technology: size does not matter; quality control of targeted clones does

Cellular & Molecular Biology Letters ◽

10.1515/cmble-2015-0047 ◽

2015 ◽

Vol 20 (5) ◽

Cited By ~ 4

Author(s):

Silvia Petrezselyova ◽

Slavomir Kinsky ◽

Dominika Truban ◽

Radislav Sedlacek ◽

Ingo Burtscher ◽

...

Keyword(s):

Quality Control ◽

Dna Sequences ◽

Genome Engineering ◽

Southern Hybridization ◽

Embryonic Stem ◽

Fluorescent Reporter ◽

Exogenous Dna ◽

Knock Out ◽

Endogenous Genes ◽

Pcr Method

AbstractClustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology has brought rapid progress in mammalian genome editing (adding, disrupting or changing the sequence of specific sites) by increasing the frequency of targeted events. However, gene knock-in of DNA cassettes by homologous recombination still remains difficult due to the construction of targeting vectors possessing large homology arms (from 2 up to 5 kb). Here, we demonstrate that in mouse embryonic stem cells the combination of CRISPR/Cas9 technology and targeting vectors with short homology arms (~ 0.3 kb) provides sufficient specificity for insertion of fluorescent reporter cassettes into endogenous genes with similar efficiency as those with large conventional vectors. Importantly, we emphasize the necessity of thorough quality control of recombinant clones by combination of the PCR method, Southern hybridization assay and sequencing to exclude undesired mutations. In conclusion, our approach facilitates programmed integration of exogenous DNA sequences at a target locus and thus could serve as a basis for more sophisticated genome engineering approaches, such as generation of reporters and conditional knock-out alleles.

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New and TALENted Genome Engineering Toolbox

Circulation Research ◽

10.1161/circresaha.113.301765 ◽

2013 ◽

Vol 113 (5) ◽

pp. 571-587 ◽

Cited By ~ 33

Author(s):

Jarryd M. Campbell ◽

Katherine A. Hartjes ◽

Timothy J. Nelson ◽

Xiaolei Xu ◽

Stephen C. Ekker

Keyword(s):

Cardiovascular Disease ◽

Gene Editing ◽

Genome Engineering ◽

Genetic Alterations ◽

Model Systems ◽

Patient Specific ◽

Specific Gene ◽

Genetic Defects ◽

Throughput Model ◽

Efficient Gene

Recent advances in the burgeoning field of genome engineering are accelerating the realization of personalized therapeutics for cardiovascular disease. In the postgenomic era, sequence-specific gene-editing tools enable the functional analysis of genetic alterations implicated in disease. In partnership with high-throughput model systems, efficient gene manipulation provides an increasingly powerful toolkit to study phenotypes associated with patient-specific genetic defects. Herein, this review emphasizes the latest developments in genome engineering and how applications within the field are transforming our understanding of personalized medicine with an emphasis on cardiovascular diseases.

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Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽

10.3390/biology10060530 ◽

2021 ◽

Vol 10 (6) ◽

pp. 530

Author(s):

Marlo K. Thompson ◽

Robert W. Sobol ◽

Aishwarya Prakash

Keyword(s):

Dna Repair ◽

Mammalian Cells ◽

Genome Stability ◽

Gene Editing ◽

Adaptive Immune System ◽

Zinc Finger Nucleases ◽

Specific Gene ◽

Target Sequence ◽

Transcription Activator ◽

Low Cytotoxicity

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

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High-throughput vectors for efficient gene silencing in plants

Functional Plant Biology ◽

10.1071/fp02033 ◽

2002 ◽

Vol 29 (10) ◽

pp. 1217 ◽

Cited By ~ 99

Author(s):

Chris A. Helliwell ◽

S. Varsha Wesley ◽

Anna J. Wielopolska ◽

Peter M. Waterhouse

Keyword(s):

Gene Silencing ◽

Insertional Mutagenesis ◽

Single Step ◽

Plant Genome ◽

Specific Gene ◽

Single Polymerase Chain Reaction ◽

Efficient Gene ◽

Gene Redundancy ◽

Endogenous Genes ◽

Rapid Generation

A major challenge in the post-genome era of plant biology is to determine the functions of all genes in the plant genome. A straightforward approach to this problem is to reduce or knockout expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study but is limited by gene redundancy, lethal knockouts, non-tagged mutants, and the inability to target the inserted element to a specific gene. The efficacy of gene silencing in plants using inverted-repeat transgene constructs that encode a hairpin RNA (hpRNA) has been demonstrated by a number of groups, and has several advantages over insertional mutagenesis. In this paper we describe two improved pHellsgate vectors that facilitate rapid generation of hpRNA-encoding constructs. pHellsgate 4 allows the production of an hpRNA construct in a single step from a single polymerase chain reaction product, while pHellsgate 8 requires a two-step process via an intermediate vector. We show that these vectors are effective at silencing three endogenous genes in Arabidopsis, FLOWERING LOCUS C, PHYTOENE DESATURASE and ETHYLENE INSENSITIVE 2. We also show that a construct of sequences from two genes silences both genes.

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Allele-specific gene editing rescues pathology in a human model of Charcot-Marie-Tooth disease type 2E

10.1101/2021.06.09.447796 ◽

2021 ◽

Author(s):

Carissa M. Feliciano ◽

Kenneth Wu ◽

Hannah L. Watry ◽

Chiara B.E. Marley ◽

Gokul N. Ramadoss ◽

...

Keyword(s):

Motor Neurons ◽

Light Chain ◽

Gene Editing ◽

Human Model ◽

Specific Gene ◽

Neurofilament Light Chain ◽

Neurofilament Light ◽

Light Chain Gene ◽

Charcot Marie Tooth ◽

Allele Specific

Many neuromuscular disorders are caused by dominant missense mutations that lead to dominant-negative or gain-of-function pathology. This category of disease is challenging to address via drug treatment or gene augmentation therapy because these strategies may not eliminate the effects of the mutant protein or RNA. Thus, effective treatments are severely lacking for these dominant diseases, which often cause severe disability or death. The targeted inactivation of dominant disease alleles by gene editing is a promising approach with the potential to completely remove the cause of pathology with a single treatment. Here, we demonstrate that allele-specific CRISPR gene editing in a human model of axonal Charcot-Marie-Tooth (CMT) disease rescues pathology caused by a dominant missense mutation in the neurofilament light chain gene (NEFL, CMT type 2E). We utilized a rapid and efficient method for generating spinal motor neurons from human induced pluripotent stem cells (iPSCs) derived from a patient with CMT2E. Diseased motor neurons recapitulated known pathologic phenotypes at early time points of differentiation, including aberrant accumulation of neurofilament light chain protein in neuronal cell bodies. We selectively inactivated the disease NEFL allele in patient iPSCs using Cas9 enzymes to introduce a frameshift at the pathogenic N98S mutation. Motor neurons carrying this allele-specific frameshift demonstrated an amelioration of the disease phenotype comparable to that seen in an isogenic control with precise correction of the mutation. Our results validate allele-specific gene editing as a therapeutic approach for CMT2E and as a promising strategy to silence dominant mutations in any gene for which heterozygous loss-of-function is well tolerated. This highlights the potential for gene editing as a therapy for currently untreatable dominant neurologic diseases.

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Enhanced Cas12a multi-gene regulation using a CRISPR array separator

10.1101/2021.01.27.428408 ◽

2021 ◽

Author(s):

Jens P. Magnusson ◽

Antonio R. Rios ◽

Lingling Wu ◽

Lei S. Qi

Keyword(s):

Mammalian Cells ◽

Gene Editing ◽

Gc Content ◽

Gene Control ◽

Naturally Occurring ◽

Crispr Array ◽

Endogenous Genes ◽

Simultaneous Activation ◽

Type V ◽

Array Performance

AbstractThe type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple crRNAs. Here, we report that Cas12a array performance is hypersensitive to the GC content of crRNA spacers, as high-GC spacers can impair activity of the downstream crRNA. We analyzed naturally occurring CRISPR arrays and observed that repeats always contain an AT-rich fragment that separates crRNAs; we term this fragment a CRISPR separator. Inspired by this observation, we designed short, AT-rich synthetic separators (synSeparators) that successfully removed the disruptive effects between crRNAs. We demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously unknown feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.

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Pluripotent stem cell SOX9 and INS reporters facilitate differentiation into insulin-producing cells

10.1101/2021.02.02.429390 ◽

2021 ◽

Author(s):

Rabea Dettmer ◽

Isabell Niwolik ◽

Ilir Mehmeti ◽

Anne Jörns ◽

Ortwin Naujok

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Beta Cells ◽

Pluripotent Stem Cells ◽

Reporter Genes ◽

Human Pluripotent Stem Cells ◽

Endogenous Gene ◽

Pancreatic Progenitors ◽

Insulin Producing Cells ◽

Endogenous Genes

AbstractDifferentiation of human pluripotent stem cells into insulin-producing stem cell-derived beta cells harbors great potential for research and therapy of diabetes. The SOX9 gene plays a crucial role during development of the pancreas and particularly in the development of insulin-producing cells as SOX9+ cells form the source for NEUROG3+ endocrine progenitor cells. For the purpose of easy monitoring of differentiation efficiencies into pancreatic progenitors and insulin-producing cells, we generated new reporter lines by knocking in a P2A-H-2Kk-F2A-GFP2 reporter genes into the SOX9 locus and a P2A-mCherry reporter gene into the INS locus mediated by CRISPR/CAS9-technology. The knock-ins enable co-expression of the endogenous genes and reporter genes, report the endogenous gene expression and enable the purification of pancreatic progenitors and insulin-producing cells using FACS or MACS. Using these cell lines we established a new differentiation protocol geared towards SOX9+ cells to efficiently drive human pluripotent stem cells into glucose-responsive beta cells.

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