Increase Editing Efficiency Research Articles

Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope

BackgroundNme2ABE8e has been constructed and characterized as a compact, accurate adenine base editor with a less restrictive dinucleotide protospacer-adjacent motif (PAM: N4CC) but low editing efficiency at challenging loci in human cells. Here, we engineered a subset of domain-inlaid Nme2Cas9 base editors to bring the deaminase domain closer to the nontarget strand to improve editing efficiency.ResultsOur results demonstrated that Nme2ABE8e-797 with adenine deaminase inserted between amino acids 797 and 798 has a significantly increased editing efficiency with a wide editing window ranging from 4 to 18 bases in mammalian cells, especially at the sites that were difficult to edit by Nme2ABE8e. In addition, by swapping the PAM-interacting domain of Nme2ABE8e-797 with that of SmuCas9 or introducing point mutations of eNme2-C in Nme2ABE8e-797, we created Nme2ABE8e-797Smu and Nme2ABE8e-797−C, respectively, which exhibited robust activities at a wide range of sites with N4CN PAMs in human cells. Moreover, the modified domain-inlaid Nme2ABE8e can efficiently restore or install disease-related loci in Neuro-2a cells and mice.ConclusionsThese novel Nme2ABE8es with increased on-target DNA editing and expanded PAM compatibility will expand the base editing toolset for efficient gene modification and therapeutic applications.

Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two

BackgroundIn plant genome editing, RNA-guided nucleases such as Cas9 from Streptococcus pyogenes (SpCas9) predominantly induce small insertions or deletions at target sites. This can be used for inactivation of protein-coding genes by frame shift mutations. However, in some cases, it may be advantageous to delete larger chromosomal segments. This is achieved by simultaneously inducing double strand breaks upstream and downstream of the segment to be deleted. Experimental approaches for the deletion of larger chromosomal segments have not been systematically evaluated.ResultsWe designed three pairs of guide RNAs for deletion of a ~ 2.2 kb chromosomal segment containing the Arabidopsis WRKY30 locus. We tested how the combination of guide RNA pairs and co-expression of the exonuclease TREX2 affect the frequency of wrky30 deletions in editing experiments. Our data demonstrate that compared to one pair of guide RNAs, two pairs increase the frequency of chromosomal deletions. The exonuclease TREX2 enhanced mutation frequency at individual target sites and shifted the mutation profile towards larger deletions. However, TREX2 did not elevate the frequency of chromosomal segment deletions.ConclusionsMultiplex editing with at least two pairs of guide RNAs (four guide RNAs in total) elevates the frequency of chromosomal segment deletions at least at the AtWRKY30 locus, and thus simplifies the selection of corresponding mutants. Co-expression of the TREX2 exonuclease can be used as a general strategy to increase editing efficiency in Arabidopsis without obvious negative effects.

Enhancement of a prime editing system via optimal recruitment of the pioneer transcription factor P65

Prime editing is a versatile gene editing tool that enables precise sequence changes of all types in the genome, but its application is rather limited by the editing efficiency. Here, we first apply the Suntag system to recruit the transcription factor P65 and enhance the desired editing outcomes in the prime editing system. Next, MS2 hairpins are used to recruit MS2-fused P65 and confirmed that the recruitment of the P65 protein could effectively improve the prime editing efficiency in both the PE3 and PE5 systems. Moreover, this suggests the increased editing efficiency is most likely associated with the induction of chromatin accessibility change by P65. In conclusion, we apply different systems to recruit P65 and enhance the prime editing efficiency of various PE systems. Furthermore, our work provides a variety of methods to work as protein scaffolds for screening target factors and thus supports further optimization of prime editing systems.

Genome editing in rice: New paths for an old crop

Abstract Rice is one of the most consumed cereals worldwide and feeds about 3 billion people. However, the occurrence of climate changes has influenced abiotic and biotic stresses and consequently affected the production and quality of rice grains. The development of new, higher yielding cultivars is necessary to ensure global food security. Although great progress has already been achieved by conventional breeding, biotechnological tools, such as transgenics and genome editing, can aid in meeting future demands. Gene editing is characterized by cutting and modifying target genes. Among the genome editing techniques, CRISPR/Cas (clustered regularly interspaced short palindromic repeats/Cas), has been proposed because of its ease of manipulation. Variants such as multiple Cas proteins, base editing and prime editing, which aim to increase editing efficiency have also been proposed. Edited plants are more accepted because they are transgene-free. Thus, the objective of this review is to describe the different gene editing techniques and their respective applications in rice breeding. It was observed that the CRISPR/Cas tool was efficient in gene editing in studies related to yield, tolerance to biotic and abiotic stresses, and rice grain quality. However, the impact of this approach in breeding programs depends on the cultivation of edited plants on a large scale in the field. Based on the progress made so far, it is believed that rice breeders can bet on CRISPR/Cas and its variants to help face the current and future challenges of climate change and high food demand.

Nucleoside analogs in ADAR guide strands targeting 5'-UA̲ sites.

Adenosine deaminases that act on RNA (ADARs) can be directed to predetermined sites in transcriptomes by forming duplex structures with exogenously delivered guide RNAs (gRNAs). They can then catalyze the hydrolytic deamination of adenosine to inosine in double stranded RNA, which is read as guanosine during translation. High resolution structures of ADAR2-RNA complexes revealed a unique conformation for the nucleotide in the guide strand base paired to the editing site's 5' nearest neighbor (-1 position). Here we describe the effect of 16 different nucleoside analogs at this position in a gRNA that targets a 5'-UA̲-3' site. We found that several analogs increase editing efficiency for both catalytically active human ADARs. In particular, 2'-deoxynebularine (dN) increased the ADAR1 and ADAR2 in vitro deamination rates when at the -1 position of gRNAs targeting the human MECP2 W104X site, the mouse IDUA W392X site, and a site in the 3'-UTR of human ACTB. Furthermore, a locked nucleic acid (LNA) modification at the -1 position was found to eliminate editing. When placed -1 to a bystander editing site in the MECP2 W104X sequence, bystander editing was eliminated while maintaining on-target editing. In vitro trends for four -1 nucleoside analogs were validated by directed editing of the MECP2 W104X site expressed on a reporter transcript in human cells. This work demonstrates the importance of the -1 position of the gRNA to ADAR editing and discloses nucleoside analogs for this site that modulate ADAR editing efficiency.

Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies

Editing efficiency is pivotal for the efficacies of CRISPR-based gene therapies. We found that fusing an HMG-D domain to the N terminus of SpCas9 (named efficiency-enhanced Cas9 [eeCas9]) significantly increased editing efficiency by 1.4-fold on average. The HMG-D domain also enhanced the activities of non-NGG PAM Cas9 variants, high-fidelity Cas9 variants, smaller Cas9 orthologs, Cas9-based epigenetic regulators, and base editors in cell lines. Furthermore, we discovered that eeCas9 exhibits comparable off-targeting effects with Cas9, and its specificity could be increased through ribonucleoprotein delivery or using hairpin single-guide RNAs and high-fidelity Cas9s. The entire eeCas9 could be packaged into an adeno-associated virus vector and exhibited a 1.7- to 2.6-fold increase in editing efficiency targeting the Pcsk9 gene in mice, leading to a greater reduction of serum cholesterol levels. Moreover, the efficiency of eeA3A-BE3 also surpasses that of A3A-BE3 in targeting the promoter region of γ-globin genes or BCL11A enhancer in human hematopoietic stem cells to reactivate γ-globin expression for the treatment of β-hemoglobinopathy. Together, eeCas9 and its derivatives are promising editing tools that exhibit higher activity and therapeutic efficacy for both invivo and exvivo therapeutics.

ADAR activation by inducing a syn conformation at guanosine adjacent to an editing site.

ADARs (adenosine deaminases acting on RNA) can be directed to sites in the transcriptome by complementary guide strands allowing for the correction of disease-causing mutations at the RNA level. However, ADARs show bias against editing adenosines with a guanosine 5' nearest neighbor (5'-GA sites), limiting the scope of this approach. Earlier studies suggested this effect arises from a clash in the RNA minor groove involving the 2-amino group of the guanosine adjacent to an editing site. Here we show that nucleosides capable of pairing with guanosine in a syn conformation enhance editing for 5'-GA sites. We describe the crystal structure of a fragment of human ADAR2 bound to RNA bearing a G:G pair adjacent to an editing site. The two guanosines form a Gsyn:Ganti pair solving the steric problem by flipping the 2-amino group of the guanosine adjacent to the editing site into the major groove. Also, duplexes with 2'-deoxyadenosine and 3-deaza-2'-deoxyadenosine displayed increased editing efficiency, suggesting the formation of a Gsyn:AH+anti pair. This was supported by X-ray crystallography of an ADAR complex with RNA bearing a G:3-deaza dA pair. This study shows how non-Watson-Crick pairing in duplex RNA can facilitate ADAR editing enabling the design of next generation guide strands for therapeutic RNA editing.

Pioneer Factor Improves CRISPR‐Based C‐To‐G and C‐To‐T Base Editing

Base editing events in eukaryote require a compatible chromatin environment, but there is little research on how chromatin factors contribute to the editing efficiency or window. By engineering BEs (base editors) fused with various pioneer factors, the authors found that SOX2 substantially increased the editing efficiency for GBE and CBE. While SoxN‐GBE (SOX2‐NH3‐GBE) improved the editing efficiency at overall cytosines of the protospacer, SoxM‐GBE/CBE (SOX2‐Middle‐GBE/CBE) enabled the higher base editing at PAM‐proximal cytosines. By separating functional domains of SOX2, the SadN‐GBE (SOX2 activation domain‐NH3‐GBE) is constructed for higher editing efficiency and SadM‐CBE for broader editing window to date. With the DNase I assay, it is also proved the increased editing efficiency is most likely associated with the induction of chromatin accessibility by SAD. Finally, SadM‐CBE is employed to introduce a stop codon in the proto‐oncogene MYC, at a locus rarely edited by previous editors with high efficiency. In this work, a new class of pioneer‐BEs is constructed by fusion of pioneer factor or its functional domains, which exhibits higher editing efficiency or broader editing window in eukaryote.

Expanding the Editing Window of Cytidine Base Editors With the Rad51 DNA-Binding Domain in Rice.

Recently developed base editors provide a powerful tool for plant research and crop improvement. Although a number of different deaminases and Cas proteins have been used to improve base editors the editing efficiency, and editing window are still not optimal. Fusion of a non-sequence-specific single-stranded DNA-binding domain (DBD) from the human Rad51 protein between Cas9 nickase and the deaminase has been reported to dramatically increase the editing efficiency and expand the editing window of base editors in the mammalian cell lines and mouse embryos. We report the use of this strategy in rice, by fusing a rice codon-optimized human Rad51 DBD to the cytidine base editors AncBE4max, AncBE4max-NG, and evoFERNY. Our results show that the addition of Rad51 DBD did not increase editing efficiency in the major editing window but the editing range was expanded in all the three systems. Replacing the human Rad51 DBD with the rice Rad51 DBD homolog also expanded the editing window effectively.

Targeting DNA polymerase to DNA double-strand breaks reduces DNA deletion size and increases templated insertions generated by CRISPR/Cas9.

Most insertions or deletions generated by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) endonucleases are short (<25 bp), but unpredictable on-target long DNA deletions (>500 bp) can be observed. The possibility of generating long on-target DNA deletions poses safety risks to somatic genome editing and makes the outcomes of genome editing less predictable. Methods for generating refined mutations are desirable but currently unavailable. Here, we show that fusing Escherichia coli DNA polymerase I or the Klenow fragment to Cas9 greatly increases the frequencies of 1-bp deletions and decreases >1-bp deletions or insertions. Importantly, doing so also greatly decreases the generation of long deletions, including those >2 kb. In addition, templated insertions (the insertion of the nucleotide 4 nt upstream of the protospacer adjacent motif) were increased relative to other insertions. Counteracting DNA resection was one of the mechanisms perturbing deletion sizes. Targeting DNA polymerase to double-strand breaks did not increase off-targets or base substitution rates around the cleavage sites, yet increased editing efficiency in primary cells. Our strategy makes it possible to generate refined DNA mutations for improved safety without sacrificing efficiency of genome editing.

CRISPR/Cas-Mediated Resistance against Viruses in Plants.

CRISPR/Cas9 provides a robust and widely adaptable system with enormous potential for genome editing directed towards generating useful products. It has been used extensively to generate resistance against viruses infecting plants with more effective and prolonged efficiency as compared with previous antiviral approaches, thus holding promise to alleviate crop losses. In this review, we have discussed the reports of CRISPR/Cas-based virus resistance strategies against plant viruses. These strategies include approaches targeting single or multiple genes (or non-coding region) in the viral genome and targeting host factors essential for virus propagation. In addition, the utilization of base editing has been discussed to generate transgene-free plants resistant to viruses. This review also compares the efficiencies of these approaches. Finally, we discuss combinatorial approaches, including multiplexing, to increase editing efficiency and bypass the generation of escape mutants.

Internally inlaid SaCas9 base editors enable window specific base editing.

Rationale: Base editors composed of catalytic defective Cas9 and cytosine or adenosine deaminase are powerful tools to convert bases in a genome. However, the fixed and narrow editing window of current base editors has impeded their utility. To increase the scope and diversify the editing patterns is quite necessary.Methods and Results: We designed a subset of base editors derived from SaCas9 in which deaminase was inlaid into various locations of the SaCas9 protein. The resulting base editors were characterized with multiple genomic sites and were found to have distinct editing features to the N-terminal SaCas9 CBE (Sa-CBE-N). Among them, Sa-CBE-693, in which a cytosine deaminase was inserted between amino acids 693 and 694, showed an increased editing efficiency and a significantly expanded editing window ranging from bases 2-18. This feature enhanced the editing efficiency of BCL11A enhancer that contains multiple consensus bases in a 15-bp fragment. Another variant, Sa-CBE-125, displayed backward-shifted editing window, which we showed was particularly powerful in editing cytosines that were accompanied with unintended bystander cytosines at their 5' side. Additionally, these editors showed reduced Cas9 independent DNA off-target editing compared with Sa-CBE-N.Conclusion: Our inlaid base editors improved the targeting scope and diversified the editing pattern.

AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines

Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, “AsCas12a Ultra”, that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.

Fertility recovery of wheat male sterility controlled by Ms2 using CRISPR/Cas9.

Fertility recovery of wheat male sterility controlled by Ms2 using CRISPR/Cas9.

Increasing CRISPR Efficiency and Measuring Its Specificity in HSPCs Using a Clinically Relevant System

Genome editing of human cluster of differentiation 34+ (CD34+) hematopoietic stem and progenitor cells (HSPCs) holds great therapeutic potential. This study aimed to optimize on-target, ex vivo genome editing using the CRISPR-Cas9 system in CD34+ HSPCs and to create a clear workflow for precise identification of off-target effects. Modified synthetic guide RNAs (gRNAs), either 2-part gRNA or single-guide RNA (sgRNA), were delivered to CD34+ HSPCs as part of ribonucleoprotein (RNP) complexes, targeting therapeutically relevant genes. The addition of an Alt-R electroporation enhancer (EE), a short, single-stranded oligodeoxynucleotide (ssODN), significantly increased editing efficiency in CD34+ HSPCs. Notably, similar editing improvement was observed when excess gRNA over Cas9 protein was used, providing a DNA-free alternative suitable for therapeutic applications. Furthermore, we demonstrated that sgRNA may be preferable over 2-part gRNA in a locus-specific manner. Finally, we present a clear experimental framework suitable for the unbiased identification of bona fide off-target sites by Genome-Wide, Unbiased Identification of Double-Strand Breaks (DSBs) Enabled by Sequencing (GUIDE-seq), as well as subsequent editing quantification in CD34+ HSPCs using rhAmpSeq. These findings may facilitate the implementation of genome editing in CD34+ HSPCs for research and therapy and can be adapted for other hematopoietic cells.

Developing high-efficiency base editors by combining optimized synergistic core components with new types of nuclear localization signal peptide

The clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas) system has been widely used for genome editing. In this system, the cytosine base editor (CBE) and adenine base editor (ABE) allow generating precise and irreversible base mutations in a programmable manner and have been used in many different types of cells and organisms. However, their applications are limited by low editing efficiency at certain genomic target sites or at specific target cytosine (C) or adenine (A) residues. Using a strategy of combining optimized synergistic core components, we developed a new multiplex super-assembled ABE (sABE) in rice that showed higher base-editing efficiency than previously developed ABEs. We also designed a new type of nuclear localization signal (NLS) comprising a FLAG epitope tag with four copies of a codon-optimized NLS (F4NLSr2) to generate another ABE named F4NLS-sABE. This new NLS increased editing efficiency or edited additional A at several target sites. A new multiplex super-assembled CBE (sCBE) and F4NLSr2 involved F4NLS-sCBE were also created using the same strategy. F4NLS-sCBE was proven to be much more efficient than sCBE in rice. These optimized base editors will serve as powerful genome-editing tools for basic research or molecular breeding in rice and will provide a reference for the development of superior editing tools for other plants or animals.

Endogenous CRISPR-Cas System-Based Genome Editing and Antimicrobials: Review and Prospects.

CRISPR-Cas systems adapt “memories” via spacers from viruses and plasmids to develop adaptive immunity against mobile genetic elements. Mature CRISPR RNAs guide CRISPR-associated nucleases to site-specifically cleave target DNA or RNA, providing an efficient genome engineering tool for organisms of all three kingdoms. Cas9, Cas12, and Cas13 are single proteins with multiple domains that are the most widely used CRISPR nucleases of the Class 2 system. However, these CRISPR endonucleases are large in size, leading to difficulty for manipulation and toxicity for cells. Most archaeal genomes and half of the bacterial genomes encode different types of CRISPR-Cas systems. Therefore, developing endogenous CRISPR-Cas systems-based genome editing will simplify manipulations and increase editing efficiency in prokaryotic cells. Here, we review the current applications and discuss the prospects of using endogenous CRISPR nucleases for genome engineering and CRISPR-based antimicrobials.

Structural Pattern Differences in Unbranched Rod-like RNA of Hepatitis Delta Virus affect RNA Editing.

Hepatitis delta virus (HDV) RNA forms an unbranched rod-like structure and complexes with the delta antigen (HDAg). Host ADAR1-catalyzed RNA editing at the amber/W site of the small HDAg leads to the production of the large HDAg, which inhibits replication and is required for virion assembly. For HDV genotype 1, amber/W editing is controlled by HDAg and the RNA structure immediate vicinity and downstream of the editing site. Here, the effects of 20 mutants carrying an increased length of consecutive base-pairing at various sites in HDV RNA on amber/W site editing were examined. All nine mutants carrying genomic regions that formed up to 15 consecutive base pairs, which is also the maximum length observed in 41 naturally occurring HDV genomes, showed normal editing rate. However, mutants carrying a 16 or 17 consecutive base-paired antigenomic segment located as far as 114 nt upstream could increase editing efficiency, possibly by interfering with HDAg binding. These data show for the first time that extended base-pairing upstream of the amber/W site could increase HDV RNA editing efficiency. Furthermore, it appears that the naturally occurring HDV RNA structures have been selected for suboptimal amber/W RNA editing, which favors the HDV replication cycle.

Application and future perspective of CRISPR/Cas9 genome editing in fruit crops

Fruit crops, including apple, orange, grape, banana, strawberry, watermelon, kiwifruit and tomato, not only provide essential nutrients for human life but also contribute to the major agricultural output and economic growth of many countries and regions in the world. Recent advancements in genome editing provides an unprecedented opportunity for the genetic improvement of these agronomically important fruit crops. Here, we summarize recent reports of applying CRISPR/Cas9 to fruit crops, including efforts to reduce disease susceptibility, change plant architecture or flower morphology, improve fruit quality traits, and increase fruit yield. We discuss challenges facing fruit crops as well as new improvements and platforms that could be used to facilitate genome editing in fruit crops, including dCas9-base-editing to introduce desirable alleles and heat treatment to increase editing efficiency. In addition, we highlight what we see as potentially revolutionary development ranging from transgene-free genome editing to de novo domestication of wild relatives. Without doubt, we now see only the beginning of what will eventually be possible with the use of the CRISPR/Cas9 toolkit. Efforts to communicate with the public and an emphasis on the manipulation of consumer-friendly traits will be critical to facilitate public acceptance of genetically engineered fruits with this new technology.

Base Editing: Chemistry on a Target Nucleotide in the Genome of Living Cells

Point mutations represent the majority of known human genetic variants associated with disease but are difficult to correct cleanly and efficiently using standard nuclease‐based genome editing methods. In this lecture I will describe the development, application, and evolution of base editing, a new approach to genome editing that directly converts a target base pair to another base pair in living cells without requiring DNA backbone cleavage or donor DNA templates. Through a combination of protein engineering and protein evolution, we recently developed two classes of base editors (BE4 and ABE) that together enable all four types of transition mutations (C to T, T to C, A to G, and G to A) to be efficiently and cleanly installed or corrected at target positions in genomic DNA. The four transition mutations collectively account for most known human pathogenic point mutations. Base editing has been widely used by many laboratories around the world in a wide range of organisms including bacteria, fungi, plants, fish, frogs, insects, mammals, and even human embryos. We have recently expanded the scope of base editing by enhancing its efficiency, product purity, targeting scope, and DNA specificity. By optimizing base editor expression, we developed “max” versions of cytosine and adenine base editors with greatly increase editing efficiency in mammalian cells. We also show that base editing can function in vivo in post‐mitotic somatic cells that do not support homology‐directed repair. To improve the targeting scope of base editing, we used our phage‐assisted continuous evolution (PACE) system to rapidly evolve Cas9 and base editor variants with both broadened PAM compatibility and higher DNA specificity. Finally, we integrated several of these developments to address cell and animal models of human genetic disease. Base editing can be used to correct pathogenic point mutations, introduce disease‐suppressing mutations, and create new models of genetic diseases.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.