Efficient Genome Editing Tool Research Articles

Application of an endogenous pGhαGloA promoter in CRISPR/Cas12a system for efficient genome editing to creat glandless cotton germplasm

The efficient genome editing tool (the CRISPR/Cas12a system) has been used in research on plant functionional genomics and improvement of agronomic traits. In this study, CRISPR/Cas12a system was optimized by using the endogenous pGhαGloA promoter in cotton. Using this system, crRNAs was driven by the Pol II pGhaGloA promoter to construct the pGhRBE3-pGhαGloA-GhPGF vector and carry out genetic transformation. The vector could work efficiently in all positive transgenic plants and the editing efficiency at the crRNA1 target site was up to 93.37%, and the editing efficiency of the crRNA2 target was up to 88.24%, which is significantly higher in editing efficiency of the pGhRBE3 system with Pol III promoter-Ubi 6.7 promoter, this result indicates that the Pol II promoter is more suitable for expressing multiple sgRNA or crRNA than the pol III promoter in cotton. The vector mainly generated the editing type of fragment deletion and the deletion size was in the range of 3-12 bp with the editing sites spanning at the 14th to 29th bases downstream of the protospacer adjacent motif (PAM). All the targeted mutation loci were stably inherited from T0 to T2 generation and three transgene-free lines with target site mutations of GhPGF gene were obtained and these glandless and gossypol-free/(low contents) cotton germplasm will play key role for healthy cottonseeds oil/cake production. Therefore, the CRISPR/Cas12a system driven by the pGhαGloA promoter can efficiently edit target genes in cotton, which provides a powerful tool for cotton functionional genomics and genetic improvement.

Genome editing tools based improved applications in macrofungi.

Macrofungi commonly referred to as Mushrooms are distributed worldwide and well known for their nutritional, medicinal, and organoleptic properties. Strain improvement in mushrooms is lagging due to paucity of efficient genome modification techniques. Thus, for advanced developments in research and commercial or economical viability and benefit, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) emerged as an efficient genome editing tool. The higher efficiency and precision of the desired genetic modification(s) are the most valuable attributes of this recent technology. The present review comprehensively summarizes various conventional methods utilized for strain improvement in mushrooms including hybridization, protoplast fusion, and di-mon mating. Furthermore, the problems associated with these techniques have been discussed besides providing the potential recluses. The significance of CRISPR/Cas9 strategies employed for improvement in various mushroom genera has been deliberated, as these strategies will paves the way forward for obtaining improved strain and effective cultivation methods for enhancing the yield and quality of the fruit bodies.

Optimizing Recombinant Cas9 Expression: Insights from E. coli BL21(DE3) Strains for Enhanced Protein Purification and Genome Editing.

The CRISPR-Cas9 system is a revolutionary tool in genetic engineering, offering unprecedented precision and efficiency in genome editing. Cas9, an enzyme derived from bacteria, is guided by RNA to edit DNA sequences within cells precisely. However, while CRISPR-Cas9 presents notable benefits and encouraging outcomes as a molecular tool and a potential therapeutic agent, the process of producing and purifying recombinant Cas9 protein remains a formidable hurdle. In this study, we systematically investigated the expression of recombinant SpCas9-His in four distinct Escherichia coli (E. coli) strains (Rosetta2, BL21(DE3), BL21(DE3)-pLysS, and BL21(DE3)-Star). Through optimization of culture conditions, including temperature and post-induction time, the BL21(DE3)-pLysS strain demonstrated efficient SpCas9 protein expression. This study also presents a detailed protocol for the purification of recombinant SpCas9, along with detailed troubleshooting tips. Results indicate successful SpCas9 protein expression using E. coli BL21(DE3)-pLysS at 0.5 mM IPTG concentration. Furthermore, the findings suggest potential avenues for further enhancements, paving the way for large-scale Cas9 production. This research contributes valuable insights into optimizing E. coli strains and culture conditions for enhanced Cas9 expression, offering a step forward in the development of efficient genome editing tools and therapeutic proteins.

An Efficient Vector-Based CRISPR/Cas9 System in Zebrafish Cell Line.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely applied in animals as an efficient genome editing tool. However, the technique is difficult to implement in fish cell lines partially due to the lack of efficient promoters to drive the expression of both sgRNA and the Cas9 protein within a single vector. In this study, it was indicated that the zebrafish U6 RNA polymerase III (ZFU6) promoter could efficiently induce tyrosinase (tyr) gene editing and lead to loss of retinal pigments when co-injection with Cas9 mRNA in zebrafish embryo. Furthermore, an optimized all-in-one vector for expression of the CRISPR/Cas9 system in the zebrafish fibroblast cell line (PAC2) was constructed by replacing the human U6 promoter with ZFU6 promoter, basing on the lentiCRISPRV2 system that widely applied in mammal cells. This new vector could successfully target the cellular communication network factor 2a (ctgfa) gene and demonstrated its function in the PAC2 cell. Notably, the vector could also be used to edit the endogenous EMX1 gene in the mammal 293T cell line, implying its wide application potential. In conclusion, we established a new gene editing tool for zebrafish cell line, which could be a useful in vitro platform for high-throughput analyzing gene function in fish.

How to use CRISPR/Cas9 in plants: from target site selection to DNA repair.

A tool for precise, target-specific, efficient, and affordable genome editing is a dream for many researchers, from those who conduct basic research to those who use it for applied research. Since 2012, we have tool that almost fulfils such requirements; it is based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems. However, even CRISPR/Cas has limitations and obstacles that might surprise its users. In this review, we focus on the most frequently used variant, CRISPR/Cas9 from Streptococcus pyogenes, and highlight key factors affecting its mutagenesis outcomes: (i) factors affecting the CRISPR/Cas9 activity, such as the effect of the target sequence, chromatin state, or Cas9 variant, and how long it remains in place after cleavage; and (ii) factors affecting the follow-up DNA repair mechanisms including mostly the cell type and cell cycle phase, but also, for example, the type of DNA ends produced by Cas9 cleavage (blunt/staggered). Moreover, we note some differences between using CRISPR/Cas9 in plants, yeasts, and animals, as knowledge from individual kingdoms is not fully transferable. Awareness of these factors can increase the likelihood of achieving the expected results of plant genome editing, for which we provide detailed guidelines.

The RUBY reporter for visual selection in soybean genome editing.

Current systems to screen for transgenic soybeans (Glycine max) involve laborious molecular assays or the expression of fluorescent markers that are difficult to see in soybean plants. Therefore, a visual system for early screening of transgenic plants would increase the efficiency of crop improvement by genome editing. The RUBY reporter system, which consists of three genes encoding betalain biosynthetic enzymes, leading to the accumulation of purple pigment in transgenic tissue, has been employed in some plants and dikaryon fungi. Here, we assessed the RUBY reporter for visual verification during soybean transformation. We show that RUBY can be expressed in soybean, allowing for visual confirmation of transgenic events without the need for specialized equipment. Plants with visible accumulation of purple pigment in any tissue were successfully transformed, confirming the accuracy of the RUBY system as a visual indicator. We also assessed the genetic stability of the transgene across generations, which can be performed very early, using the cotyledons of the progeny. Transgene-free seedlings have a distinct green color, facilitating the selection of genome-edited but transgene-free soybean seedlings for harvest. Using the RUBY system, we quickly identified a transgene-free Gmwaxy mutant in the T1 generation. This system thus provides an efficient and convenient tool for soybean genome editing.

Genome editing in macroalgae: advances and challenges.

This minireview examines the current state and challenges of genome editing in macroalgae. Despite the ecological and economic significance of this group of organisms, genome editing has seen limited applications. While CRISPR functionality has been established in two brown (Ectocarpus species 7 and Saccharina japonica) and one green seaweed (Ulva prolifera), these studies are limited to proof-of-concept demonstrations. All studies also (co)-targeted ADENINE PHOSPHORIBOSYL TRANSFERASE to enrich for mutants, due to the relatively low editing efficiencies. To advance the field, there should be a focus on advancing auxiliary technologies, particularly stable transformation, so that novel editing reagents can be screened for their efficiency. More work is also needed on understanding DNA repair in these organisms, as this is tightly linked with the editing outcomes. Developing efficient genome editing tools for macroalgae will unlock the ability to characterize their genes, which is largely uncharted terrain. Moreover, given their economic importance, genome editing will also impact breeding campaigns to develop strains that have better yields, produce more commercially valuable compounds, and show improved resilience to the impacts of global change.

CRISPR/Cpf1–FOKI-induced gene editing in Gluconobacter oxydans

Gluconobacter oxydans is an important Gram-negative industrial microorganism that produces vitamin C and other products due to its efficient membrane-bound dehydrogenase system. Its incomplete oxidation system has many crucial industrial applications. However, it also leads to slow growth and low biomass, requiring further metabolic modification for balancing the cell growth and incomplete oxidation process. As a non-model strain, G. oxydans lacks efficient genome editing tools and cannot perform rapid multi-gene editing and complex metabolic network regulation. In the last 15 years, our laboratory attempted to deploy multiple CRISPR/Cas systems in different G. oxydans strains and found none of them as functional. In this study, Cpf1-based or dCpf1-based CRISPRi was constructed to explore the targeted binding ability of Cpf1, while Cpf1–FokI was deployed to study its nuclease activity. A study on Cpf1 found that the CRISPR/Cpf1 system could locate the target genes in G. oxydans but lacked the nuclease cleavage activity. Therefore, the CRISPR/Cpf1–FokI system based on FokI nuclease was constructed. Single-gene knockout with efficiency up to 100% and double-gene iterative editing were achieved in G. oxydans. Using this system, AcrVA6, the anti-CRISPR protein of G. oxydans was discovered for the first time, and efficient genome editing was realized.

An engineered Cas12i nuclease that is an efficient genome editing tool in animals and plants

The type V-I CRISPR-Cas system is becoming increasingly more attractive for genome editing. However, natural nucleases of this system often exhibit low efficiency, limiting their application. Here, we used structure-guided rational design and protein engineering to optimize an uncharacterized Cas12i nuclease, Cas12i3. As a result, we developed Cas-SF01, a Cas12i3 variant that exhibits significantly improved gene editing activity in mammalian cells. Cas-SF01 shows comparable or superior editing performance compared to SpCas9 and other Cas12 nucleases. Compared to natural Cas12i3, Cas-SF01 has an expanded PAM range and effectively recognizes NTTN and noncanonical NATN and TTVN PAMs. In addition, we identified an amino acid substitution, D876R, that markedly reduced the off-target effect while maintaining high on-target activity, leading to the development of Cas-SF01HiFi (high-fidelity Cas-SF01). Finally, we show that Cas-SF01 has high gene editing activities in mice and plants. Our results suggest that Cas-SF01 can serve as a robust gene editing platform with high efficiency and specificity for genome editing applications in various organisms.

Advances in transposable elements: from mechanisms to applications in mammalian genomics

It has been 70 years since Barbara McClintock discovered transposable elements (TE), and the mechanistic studies and functional applications of transposable elements have been at the forefront of life science research. As an essential part of the genome, TEs have been discovered in most species of prokaryotes and eukaryotes, and the relative proportion of the total genetic sequence they comprise gradually increases with the expansion of the genome. In humans, TEs account for about 40% of the genome and are deeply involved in gene regulation, chromosome structure maintenance, inflammatory response, and the etiology of genetic and non-genetic diseases. In-depth functional studies of TEs in mammalian cells and the human body have led to a greater understanding of these fundamental biological processes. At the same time, as a potent mutagen and efficient genome editing tool, TEs have been transformed into biological tools critical for developing new techniques. By controlling the random insertion of TEs into the genome to change the phenotype in cells and model organisms, critical proteins of many diseases have been systematically identified. Exploiting the TE’s highly efficient in vitro insertion activity has driven the development of cutting-edge sequencing technologies. Recently, a new technology combining CRISPR with TEs was reported, which provides a novel targeted insertion system to both academia and industry. We suggest that interrogating biological processes that generally depend on the actions of TEs with TEs-derived genetic tools is a very efficient strategy. For example, excessive activation of TEs is an essential factor in the occurrence of cancer in humans. As potent mutagens, TEs have also been used to unravel the key regulatory elements and mechanisms of carcinogenesis. Through this review, we aim to effectively combine the traditional views of TEs with recent research progress, systematically link the mechanistic discoveries of TEs with the technological developments of TE-based tools, and provide a comprehensive approach and understanding for researchers in different fields.

A SEVA-based, CRISPR-Cas3-assisted genome engineering approach for Pseudomonas with efficient vector curing

ABSTRACT The development of CRISPR-Cas-based engineering technologies has revolutionized the microbial biotechnology field. Over the years, the Class II Type II CRISPR-Cas9 system has become the gold standard for genome editing in many bacterial hosts. However, the Cas9 system does not allow efficient genomic integration in Pseudomonas putida , an emerging Synthetic Biology host, without the assistance of lambda-Red recombineering. In this work, we utilize the alternative Class I Type I-C CRISPR-Cas3 system from Pseudomonas aeruginosa as a highly efficient genome editing tool for P. putida and P. aeruginosa . This system consists of two vectors, one encoding the Cas genes, CRISPR array and targeting spacer, and a second Standard European Vector Architecture vector, containing the homologous repair template. Both vectors are Golden Gate compatible for rapid cloning and are available with multiple antibiotic markers, for application in various Gram-negative hosts and different designs. By employing this Cas3 system, we successfully integrated an 820-bp cassette in the genome of P. putida and performed several genomic deletions in P. aeruginosa within a week, with an efficiency of >83% for both hosts. Moreover, by introducing a universal self-targeting spacer, the Cas3 system rapidly cures all helper vectors, including itself, from the host strain in a matter of days. As such, this system constitutes a valuable engineering tool for Pseudomonas , to complement the existing range of Cas9-based editing methods, and facilitates genomic engineering efforts of this important genus. IMPORTANCE The CRISPR-Cas3 editing system as presented here facilitates the creation of genomic alterations in Pseudomonas putida and Pseudomonas aeruginosa in a straightforward manner. By providing the Cas3 system as a vector set with Golden Gate compatibility and different antibiotic markers, as well as by employing the established Standard European Vector Architecture (SEVA) vector set to provide the homology repair template, this system is flexible and can readily be ported to a multitude of Gram-negative hosts. Besides genome editing, the Cas3 system can also be used as an effective and universal tool for vector curing. This is achieved by introducing a spacer that targets the origin-of-transfer, present on the majority of established (SEVA) vectors. Based on this, the Cas3 system efficiently removes up to three vectors in only a few days. As such, this curing approach may also benefit other genomic engineering methods or remove naturally occurring plasmids from bacteria.

Heterologous Single-Strand DNA-Annealing and Binding Protein Enhance CRISPR-Based Genome Editing Efficiency in Komagataella phaffii.

The industrial yeast Komagataella phaffii is a highly effective platform for heterologous protein production, owing to its high protein expression and secretion capacity. Heterologous genes and proteins are involved in multiple processes, including transcription, translation, protein folding, modification, transportation, and degradation; however, engineering these proteins and genes is challenging due to inefficient genome editing techniques. We employed Pseudomonas aeruginosa phage single-stranded DNA-annealing protein (SSAP) PapRecT and P. aeruginosa single-stranded DNA-binding protein (SSB) PaSSB to introduce SSAP-SSB-based homology recombination, which facilitated K. phaffii CRISPR-based genome engineering. Specifically, a host-independent method was developed by expressing sgRNA with PapRecT-PaSSB in a single plasmid, with which only a 50 bp short homologous arm (HA) reached a 100% positive rate for CRISPR-based gene insertion, reaching 18 colony-forming units (CFU) per μg of donor DNA. Single deletion using 1000 bp HA attained 100%, reaching 68 CFUs per μg of donor DNA. Using this efficient CRISPR-based genome editing tool, we integrated three genes (INO4, GAL4-like, and PAB1) at three different loci for overexpression to realize the collaborative regulation of human-lactalbumin (α-LA) production. Specifically, we strengthened phospholipid biosynthesis to facilitate endoplasmic reticulum membrane formation and enhanced recombinant protein transcription and translation by overexpressing transcription and translation factors. The final production of α-LA in the 3 L fermentation reached 113.4 mg L-1, two times higher than that of the strain without multiple site gene editing, which is the highest reported titer in K. phaffii. The CRISPR-based genome editing method developed in this study is suitable for the synergistic multiple-site engineering of protein and biochemical biosynthesis pathways to improve the biomanufacturing efficiency.

CRISPR/Cas9 Editing Sites Identification and Multi-Elements Association Analysis in Camellia sinensis.

CRISPR/Cas9 is an efficient genome-editing tool, and the identification of editing sites and potential influences in the Camellia sinensis genome have not been investigated. In this study, bioinformatics methods were used to characterise the Camellia sinensis genome including editing sites, simple sequence repeats (SSRs), G-quadruplexes (GQ), gene density, and their relationships. A total of 248,134,838 potential editing sites were identified in the genome, and five PAM types, AGG, TGG, CGG, GGG, and NGG, were observed, of which 66,665,912 were found to be specific, and they were present in all structural elements of the genes. The characteristic region of high GC content, GQ density, and PAM density in contrast to low gene density and SSR density was identified in the chromosomes in the joint analysis, and it was associated with secondary metabolites and amino acid biosynthesis pathways. CRISPR/Cas9, as a technology to drive crop improvement, with the identified editing sites and effector elements, provides valuable tools for functional studies and molecular breeding in Camellia sinensis.

Hs1Cas12a and Ev1Cas12a confer efficient genome editing in plants.

Cas12a, also known as Cpf1, is a highly versatile CRISPR-Cas enzyme that has been widely used in genome editing. Unlike its well-known counterpart, Cas9, Cas12a has unique features that make it a highly efficient genome editing tool at AT-rich genomic regions. To enrich the CRISPR-Cas12a plant genome editing toolbox, we explored 17 novel Cas12a orthologs for their genome editing capabilities in plants. Out of them, Ev1Cas12a and Hs1Cas12a showed efficient multiplexed genome editing in rice and tomato protoplasts. Notably, Hs1Cas12a exhibited greater tolerance to lower temperatures. Moreover, Hs1Cas12a generated up to 87.5% biallelic editing in rice T0 plants. Both Ev1Cas12a and Hs1Cas12a achieved effective editing in poplar T0 plants, with up to 100% of plants edited, albeit with high chimerism. Taken together, the efficient genome editing demonstrated by Ev1Cas12a and Hs1Cas12a in both monocot and dicot plants highlights their potential as promising genome editing tools in plant species and beyond.

Efficient and stable CRISPR/Cas9-mediated genome-editing of human type 2 innate lymphoid cells.

Innate lymphoid cells (ILCs) are a family of innate lymphocytes with important roles in immune response coordination and maintenance of tissue homeostasis. The ILC family includes group 1 (ILC1s), group 2 (ILC2s) and group 3 (ILC3s) 'helper' ILCs, as well as cytotoxic Natural Killer (NK) cells. Study of helper ILCs in humans presents several challenges, including their low proportions in peripheral blood or needing access to rare samples to study tissue resident ILC populations. In addition, the lack of established protocols harnessing genetic manipulation platforms has limited the ability to explore molecular mechanism regulating human helper ILC biology. CRISPR/Cas9 is an efficient genome editing tool that enables the knockout of genes of interest, and is commonly used to study molecular regulation of many immune cell types. Here, we developed methods to efficiently knockout genes of interest in human ILC2s. We discuss challenges and lessons learned from our CRISPR/Cas9 gene editing optimizations using a nucleofection transfection approach and test a range of conditions and nucleofection settings to obtain a protocol that achieves effective and stable gene knockout while maintaining optimal cell viability. Using IL-4 as a representative target, we compare different ribonucleoprotein configurations, as well as assess effects of length of time in culture and other parameters that impact CRISPR/Cas9 transfection efficiency. Collectively, we detail a CRISPR/Cas9 protocol for efficient genetic knockout to aid in studying molecular mechanism regulating human ILC2s.

Efficient Genome and Base Editing in Human Cells Using ThermoCas9.

Most genetic engineering applications reported thus far rely on the type II-A CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpyCas9), limiting the genome-targeting scope. In this study, we demonstrate that a small, naturally accurate, and thermostable type II-C Cas9 ortholog from Geobacillus thermodenitrificans (ThermoCas9) with alternative target site preference is active in human cells, and it can be used as an efficient genome editing tool, especially for gene disruption. In addition, we develop a ThermoCas9-mediated base editor, called ThermoBE4, for programmable nicking and subsequent C-to-T conversions in human genomes. ThermoBE4 exhibits a three times larger window of activity compared with the corresponding SpyCas9 base editor (BE4), which may be an advantage for gene mutagenesis applications. Hence, ThermoCas9 provides an alternative platform that expands the targeting scope of both genome and base editing in human cells.

Genetic engineering strategies for sustainable polyhydroxyalkanoate (PHA) production from carbon-rich wastes

Polyhydroxyalkanoates (PHAs) are promising biopolymers for biomedical applications due to their excellent biocompatibility and biodegradability. However, the high production cost mainly resulting from the pure sugar substrate limits PHA commercialization. It makes various carbon-rich wastes potential substrates for PHA production. The integration and optimization of metabolic pathways can further enhance the conversion of carbon-rich wastes to PHAs. Genetic engineering strategies focusing on carbon flux and energy metabolism have improved the PHA production capacities of targeted strains by promoting substrate assimilation, enhancing PHA synthesis, and reducing branch metabolism. CRISPR/Cas9-based systems have also served as efficient genome editing tools to improve the efficiency of metabolic modification. Genetic modification requires fitness among strains, substrates, and products. Therefore, this review outlined various efficient genetic engineering approaches to promote PHA production and discussed their feasibility in valorizing representative carbon-rich wastes. To further illustrate the widespread applicability of metabolic modification to support microbial cell factories with core PHA production, this review also involved advanced fermentation approaches and co-production systems for PHA production by engineered strains.

The Potential Revolution of Cancer Treatment with CRISPR Technology.

Immuno-oncology (IO) and targeted therapies, such as small molecule inhibitors, have changed the landscape of cancer treatment and prognosis; however, durable responses have been difficult to achieve due to tumor heterogeneity, development of drug resistance, and adverse effects that limit dosing and prolonged drug use. To improve upon the current medicinal armamentarium, there is an urgent need for new ways to understand, reverse, and treat carcinogenesis. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) 9 is a powerful and efficient tool for genome editing that has shown significant promise for developing new therapeutics. While CRISPR/Cas9 has been successfully used for pre-clinical cancer research, its use in the clinical setting is still in an early stage of development. The purpose of this review is to describe the CRISPR technology and to provide an overview of its current applications and future potential as cancer therapies.

CRISPR-Cas12a-based genome editing and transcriptional repression for biotin synthesis in Pseudomonas mutabilis.

To establish a dual-function clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system combined genome editing and transcriptional repression for multiplex metabolic engineering of Pseudomonas mutabilis. This CRISPR-Cas12a system consisted of two plasmids that enabled single gene deletion, replacement, and inactivation with efficiency >90% for most targets within 5 days. With the guidance of truncated crRNA containing 16 bp spacer sequences, a catalytically active Cas12a could be employed to repress the expression of the reporter gene eGFP up to 66.6%. When bdhA deletion and eGFP repression were tested simultaneously by transforming a single crRNA plasmid and Cas12a plasmid, the knockout efficiency reached 77.8% and the expression of eGFP was decreased by >50%. Finally, the dual-functional system was demonstrated to increase the production of biotin by 3.84-fold, with yigM deletion and birA repression achieved simultaneously. This CRISPR-Cas12a system is an efficient genome editing and regulation tool to facilitate the construction of P. mutabilis cell factories.

Web-Based Computational Tools for Base Editors.

CRISPR-based base editors are efficient genome editing tools for use in base correction. Currently, there are various versions and types of base editors with different substitution patterns, editing windows, and protospacer adjacent motif (PAM) sequences. For the design of target sequences, consideration of off-target sequences is required. In addition, for assessment of base editing outcomes in bulk populations, the analysis of high-throughput sequencing data is required. Several web browser-based computation programs have been developed for the purpose of target design and NGS data analysis, especially for users with less computational knowledge. In this manuscript, depending on the purpose of each program, we provide an explanation of useful tools including BE-Designer for design of targets and BE-Analyzer for analysis of NGS data that were developed by our group, CRISPResso2 for analysis of NGS data developed by Luca Pinello group, DeepBaseEditor for prediction of target efficiency developed by Hyongbum Henry Kim group, and BE-Hive for prediction of target outcome developed by David Liu group.