GENOME-WIDE ASSESSMENT OF OFF-TARGET EFFECTS FOR CAS12B, MULTIPLEX CAS12A

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 1023757
• Grant No.: 2020-33522-32274
• Cumulative Award Amt.: $499,400.00
• Proposal No.: 2020-02900
• Project Start Date: Sep 1, 2020
• Project End Date: Aug 31, 2025
• Grant Year: 2020
• Program Code: [HX]- Biotechnology Risk Assessment

Recipient Organization
UNIV OF MARYLAND
(N/A)
COLLEGE PARK,MD 20742

Performing Department
Plant Science & Landscape Arch

Non Technical Summary
With the advances of gene editing technologies, it is now possible to modify a plant genome at a desired site, which is unlike conventional genetic engineering where a transgene is introduced and integrates randomly into a genome. There has been a swift increase in applications of CRISPR-Cas systems during the past few years in part due to the long-term desire for a technology that allows precise modification and is straightforward to design. Another attractive feature is that the CRISPR-Cas transgene can be genetically segregated away, leaving only the targeted modification in the progeny. Based on the recently passed SECURE rule, USDA-APHIS will not regulate gene edited crops if the introduced DNA changes can be found in nature or derived from conventional breeding methods. Science based regulation will greatly encourage the use of gene editing technologies for fast-track crop breeding to make improved crops that are nutritious, high yield, and resistant to biotic and abiotic stresses. Gene editing has also greatly stimulated innovation in plant synthetic biology and Agriculture. In this project, we propose to assess three emerging CRISPR genome editing systems for any unintended modifications at other loci in the genomes of recovered edited rice lines. We specifically address the BRAG program area "5g", where it calls for comparison of the types and frequencies of nucleic acid changes introduced into important crop genomes, via genetic engineering techniques versus other plant breeding techniques. Therefore, we will specifically assess for unintended off-target effects in the genomes of edited lines recovered from 1) Cas12b editing, 2) Cas12a based multiplexed editing, and 3) Cas9 based prime editing. In parallel, we will do a comparison with control lines recovered through the plant regeneration and transformation phases because of unintended somaclonal variations. The relevance of the proposed project to the BRAG program is very significant because our goals are closely aligned with the mission, in that the results will provide information accessible by federal regulatory agencies to consider during evaluation for introduction of CRISPR-Cas generated organisms into the environment. The knowledge generated from our work will greatly aid plant scientists practicing gene editing in crops as well as facilitate the decision-making process at regulatory agencies such as USDA and FDA.

Animal Health Component

(N/A)

Research Effort Categories

Basic

80%

Applied

(N/A)

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1530	1080	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1530 - Rice;

Field Of Science
1080 - Genetics;

Keywords

cas12b

multiplex cas12a

off-target effects

prime editor

rice

whole-genome sequencing

Goals / Objectives
Objective 1. Assess off-target effects of Cas12b in rice by whole genome sequencing (WGS) (Year 1)CRISPR (clustered regularly interspaced short palindromic repeats)-Cas12b, formerly known as C2c1, is a Class 2 Type V-B endonuclease. Cas12b recognizes target sequences with a distal 5' T-rich Protospacer Adjacent Motif (PAM) sequence and generates staggered DNA Double Strand Breaks (DSBs). CRISPR-Cas12b nuclease has further expanded the toolbox for precise plant genome editing. Our initial analysis identified AaCas12b and BhCas12b-v4 as efficient gene editing nucleases in rice. These analyses however only considered targeting specificity conferred by single guide RNAs (sgRNAs). Cas12b has different PAM preferences from commonly used Cas12a and Cas9 nucleases. Also, it is not clear about in vivo DNA binding affinity and dynamics of nuclease activity by Cas12b, as compared to Cas12a and Cas9. In Objective 1, we will apply AaCas12b and BhCas12b-v4 to edit three target sites T0 plants in Kitaake, a Japonica rice variety. The edited rice plants, along with controls plants such as Cas12b vector-only transgenic plants, tissue-culture only regenerated plants, and parental wild-type plants, will be subjected for whole genome sequencing (WGS). By analyzing WGS data, we expect to reveal any potential off-target effects caused by either AaCas12b or BhCas12b-v4. With such genome-wide off-target analysis, we expect to benchmark both Cas12b nucleases as precise genome editing tools in plants.Objective 2: Assess off-target effects of a highly efficient multiplex Cas12a system in rice by WGS (Years 1 and 2)Cas12a (formerly known as Cpf1, CRISPR from Prevotella and Francisella 1), a Class 2 type V-A endonuclease, is an RNA-guided nuclease that is distinct from Cas9. First, the TTTV (V=A, C, G) PAM requirement allows Cas12a to target thymidine-rich regions. Second, Cas12a only requires a short crRNA (~42 nt), making Cas12a crRNA easy to synthesize, multiplex and engineer. Third, Cas12a also possesses RNase activity, which is able to process a CRISPR array for multiplexed genome editing. Fourth, Cas12a generates a DSB with staggered ends distal from the PAM site, which allows continuous cleavage of DNA and may promote NHEJ-based gene insertion. In recently, highly efficient gene editing with Cas12a has been demonstrated in a wide collection of plant species, including Arabidopsis, rice, maize, wheat, tomato, cotton and citrus. However, it is unclear for its off-target effects when used in a multiplexed editing setting in plants. In Objective 2, we will assess off-target effects by a most potent multiplexed Cas12a genome editing system that we have recently developed. We will apply two potent Cas12a nucleases, LbCas12a and Mb2Cas12a, to simultaneously edit 6 and 16 target sites in the rice genome of Kitaake. The resulting multiplex edited T0 lines along will different groups of control lines will be subjected for WGS and analysis. With this comprehensive assessment, we anticipate revealing any potential off-target effects due of multiplexed editing at many loci in a plant genome. Our results will shed light on multiplexed and large-scale editing applications based on Cas12a and other Cas nucleases.Objective 3: Assess off-target effects of prime editors in rice at the genome and transcriptome levels (Years 1-3)Recently, an exciting precise genome editing technology called prime editing was developed and demonstrated in humans and plants. The so-called Prime Editor (PE) introduces sequence specific changes without DSBs or donor DNA. It is based on CRISPR-Cas9 system where Cas9H840A nickase is fused with an engineered Moloney murine leukemia virus (M-MLV) reverse transcriptase (RT) at the C-terminus. The system uses a prime editing guide RNA (pegRNA) that has an extension at 3' end to include the desired nucleotide changes and prime binding site (PBS) for annealing and initiating reverse transcription. Very recently, we and others have shown prime editing systems work in plant cells for precise gene editing. It is however unclear about potential off-target effects of the prime editing systems in plants. In Objective 3, we will investigate off-target effects of prime editing in rice. First, we will try to engineer an improved version of Plant Prime Editor 2 (P-PE2) which we term as P-PE2-h (h stands for high activity). Then, we will apply P-PE2-h to target select genes for precise gene editing. We will conduct WGS using the edited T0 plants and the control plants. In addition, we will also pursue RNA sequencing (RNAseq) to compare the transcriptomes among these plants. With these analyses, we will learn off-target effects of our prime editing system at both genome and transcriptome levels. Such knowledge will be valuable to truly benchmark prime editing technologies on targeting specificity and safety in plants.

Project Methods
Objective 1. Assess off-target effects of Cas12b in rice by whole genome sequencing (WGS) (Year 1)In Objective 1, we will apply AaCas12b and BhCas12b-v4 nucleases to edit three target sites for which we have already generated the T-DNA vectors. We will generate about 20 transgenic rice plants for each construct using Agrobacterium-mediated transformation by following our established protocol. The individual T0 lines will be genotyped by PCR amplification of the target sites, followed by Sanger sequencing and analysis with Tide and CRISPR-GE programs. Two independently edited plants for each construct will be chosen for WGS. Meanwhile, we will infect rice calli with Agrobacterium carrying the AaCas12b and Bh-Cas12b transgenes without any sgRNA. The resulting plants will be named as Cas12b-only plants. In addition, tissue culture-only plants will be generated as another type of control to assess mutations derived from the tissue culture process. Wild type (non-transgenic) plants will also be included as the control group for filtering out preexisting mutations (as compared to the reference genome) in the parental lines. We will then conduct WGS based on the Illumina HiSeq platform on 12 edited lines (2 T0 edited lines x 2 Cas12b nucleases x 3 target sites) and 8 control plants (2 wild type [WT] plants, 2 tissue culture-only plants, 2 AaCas12b only plants and 2 BhCas12b-v4 only plants). In total, there will 20 rice samples for WGS. For each sample, we will target a sequencing depth of 50x. For the rice genome of ~400Mb, we will require an average of ~23Gb reads per sample. After high quality genomic DNA is extracted from selected rice lines, DNA samples will be used for library preparation and high-throughput sequencing based on an Illumina HiSeq or similar platform. The WGS data will be analyzed for off-target mutations according to our established pipeline. Briefly, adapters of raw sequencing reads will be trimmed using SKEWER tool and the Illumina TruSeq adapter. Cleaned reads from rice will be mapped to reference sequence of rice (http://rice.plantbiology.msu.edu/).The Genome Analysis Toolkit (GATK) will be used to realign reads near indels and recalibrate base quality scores. A known single nucleotide polymorphism (SNP) and insertion/deletion (indel) database for GATK best practices will be downloaded for rice from the SNP-Seek Database (http://snp-seek.irri.org/). Whole genome SNPs or indels will be detected with three independent software programs. These mutations will be mapped to putative off-target sites that are computationally predicted, allowing for identification of true off-target mutations caused by Cas12b.Objective 2: Assess off-target effects of a highly efficient multiplex Cas12a system in rice by WGS (Years 1 and 2)In Objective 2, we will pursue 2 case studies for evaluating off-target effects with the highly efficient multiplex LbCas12a and Mb2Cas12a systems. In the first case, we will simultaneously edit 4 target sites in the rice genome with LbCas12a and Mb2Cas12a. This experiment allows for a direct comparison on editing efficiency and off-target effects of these two promising Cas12a nucleases in rice. In the second case, we will simultaneously edit 16 sites on 9 chromosomes in the rice genome with LbCas12a. This second case will allow us to simulate a high-level multiplexed genome editing application so that we can investigate genome integrity or damage (off-target effects) with many concurrently DSBs induced within a single cell or plant. To this end, we will generate three multiplex Cas12a vectors for simultaneous editing 4 and 16 sites by Mb2Cas12a and LbCas12a, respectively. These three multiplex Cas12a T-DNA expression vectors will be generated by following our established molecular cloning protocols based on Golden Gate cloning and Gateway cloning strategies. We will assess the editing efficiency for each crRNA in these vectors in rice protoplasts by deep sequencing of PCR amplicons. Upon validation, we will carry them to rice stable transformation. For each of the three constructs, we will generate about 20 T0 stable rice transformants. The individual lines will be genotyped by PCR amplification of the target sites, followed by Sanger sequencing and decoding. The analysis will identify heterozygous, homozygous and biallelic mutants for each target gene in each line. For WGS of 4-site multiplexing, we will choose 2 independent lines which possess high levels of multiplexed editing, e.g. nearly all target genes are biallelically edited. For WGS of 16-site multiplexing, we will choose 5 independent lines which are multiplex edited. This design will result in 9 edited samples (2 edited by MbCas12a at 4 sites; 2 edited by LbCas12a at 4 sites; 5 edited by LbCas12a at 16 sites). In addition, there will be 9 control plant samples (3 WT plants, 2 tissue culture-only plants, 2 Mb2Cas12a only plants, and 2 LbCas12a only plants). In total, there will be 18 rice DNA samples for WGS. We will follow a similar WGS data analysis pipeline as described in Objective 1. The data will be analyzed for off-target mutations, with specific attentions to chromosomal truncations and translocations.Objective 3: Assess off-target effects of prime editors in rice at the genome and transcriptome levels (Years 1-3)To improve prime editing in plants, we will survey a list of RNA viruses found in temperate environments to identify suitable RTs for testing in plants. We will replace M-MLV RT in P-PE3-V1 with these new RTs to make a series of P-PE2 fusion proteins as new prime editors. They will be tested in rice protoplasts by targeting the 4 chosen target sites in OsALS and OsEPSPS for introducing herbicide resistance alleles. The editing results will be assessed by deep sequencing of PCR amplicons. The best version that shows much higher editing efficiency will be identified and named as P-PE2-h (h stands for high activity). With P-PE2-h, we hope to obtain ~5% or higher prime editing frequencies at all target sites in rice protoplasts. Then, we will carry out stable rice transformation with the best vector of optimized P-PE2-h and pegRNA combination for the targets in OsALS and OsEPSPS. For each of the four T-DNA constructs, about 50 transgenic T0 lines will be obtained for genotyping based on Sanger sequencing. We hope to identify more than 3 independent lines that are at least monoallelically edited by prime editing. Three edited plants for each construct, preferentially biallelically edited plants, will be chosen for WGS. There will be a total of 8 edited plants (2 independent edited lines x 4 constructs/targets). We will include 6 control plants (2 WT plants, 2 tissue culture-only plants, and 2 P-PE2-h only plants). Thus, a total of 14 plants will be subjected for WGS as well as RNAseq. We will follow a similar WGS data analysis pipeline as described in Objective 1. The analyzed data will reveal potential off-target effects of prime editing at genome and transcriptome levels, respectively.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:This project will provide cutting-edge training on plant genome editing technology development and evaluation for a post-doc, as well as multiple undergraduate and high school interns. The new CRISPR genome editing systems with improved editing activity will directly benefit many plant scientists who are interested in applying this exciting technology for foundational and applied research. The off-targeting data resulting from whole genome sequencing and RNA sequencing will guide breeders in the public sector and private industry in choosing appropriate CRISPR technologies for achieving their product development goals. The off-target information on Cas12b, multiplex Cas12a and prime editing will also aid government agencies, such as USDA and FDA, to make science-based regulatory rules and rational decisions. In this reporting period, the PI has presented the work and acknowledged this NIFA funding in the following presentations: 1. June 18, 2024. Plant Genome Stability and Change-EMBO Workshop. Olomouc, Czech Republic. 2. May 23, 2024. Plant Center Spring Symposium, University of Georgia. 3. April 16, 2024. Innovative Genomics Institute, UC-Berkeley. 4. March 7, 2024. aBiotech Journal Webinar. 5. March 5, 2024. Recombination and Genome Editing in Plants. Federation of American Societies for Experimental Biology (FASEB). 6. February 22, 2024. 6th CRISPR AgBio Congress. Theme: Engineer the Next Generation of Agriculture. 7. February 20, 2024. Enhancing the Global Food System's Resilience to Biological Threats. Scowcroft Institute of International Affairs, The BUSH School, TAMU. 8. January 30, 2024, Maximizing Agriculture to Enhance Nutrient Composition to Better Fulfill Dietary Recommendations. Workshop by National Academies of Sciences, Engineering, and Medicine. 9. January 14, 2024, Plant and Animal Genome Conference. Organellar Genome Engineering Workshop. San Diego, CA. 10. January 14, 2024, Plant and Animal Genome Conference. Development and Application of Genome Engineering and Transgenic Technology to the Agriculture Workshop. San Diego, CA. 11. October 13, 2023. Department of Plant Biology, Rutgers University, NJ. 12. September 29, 2023. School of Life Sciences, University of Nevada, Las Vegas, NV. The audience of these presentations was very diverse, including students, academic researchers, industry researchers and governmental officials from different countries. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training opportunities for three postdocs, one PhD student, one visiting PhD student, and one undergraduate intern in the Qi lab. How have the results been disseminated to communities of interest?The WGS data for off-target analysis of multiplexed Cas12a systems have been deposited to the NCBI public database. The WGS data for on and off-target analysis of multiple Cas12b systems have also been deposited to NCBI. Two manuscripts, corresponding to Objective 1 and Objective 2, have been published in peer-reviewed journals. The PI also gave presentations in several conferences and webinars (see section on Target Audience below). What do you plan to do during the next reporting period to accomplish the goals?To date, most of the experiments have been done according to our timeline. Importantly, Objectives 1 and 2 have been finished and published. Due to the COVID-19 pandemic and personnel changes, we delayed our start time for Objective 3 on developing prime editors and analyzing the off-target effects of these prime editors. Furthermore, we met some technical challenges in identifying positive rice lines that were edited by prime editing, with the PE systems that were constructed. However, we are confident that our CRISPR-Combo-based PE systems are very promising. So, we will continue to develop improved PE systems based on CRISPR-Combo and the resulting data should help us fulfill the completion of the project in one year.

Impacts
What was accomplished under these goals? CRISPR-Cas12b, multiplexed Cas12a, and Cas9-based prime editors are exciting genome editing tools that help create precise mutations in elite cultivars, hence greatly accelerating crop breeding. However, there is a key knowledge gap regarding any potential off-target effects of these three emerging new genome editing technologies. This knowledge gap can be filled by a comprehensive analysis of the edited plants through whole genome sequencing (WGS). In this project, we will use WGS to assess the potential off-target effects of these genome editing systems in rice, a major stable crop feeding nearly half of the world population. Importantly, we will assess the off-target effects of these new tools in precisely edited rice plants by WGS and by including all necessary control plants. Impact: These three new CRISPR systems have great promise in the precision breeding of resilient crops that can fight global hunger and catch up with climate change. The off-target information of these genome editing tools resulting from this project will aid regulatory agencies worldwide to guide the proper use of these new breeding technologies in a science-based framework. We specifically address the BRAG program area "5g", where it calls for a comparison of the types and frequencies of nucleic acid changes introduced into important crop genomes, via genetic engineering techniques versus other plant breeding techniques. Objective 1. Assess off-target effects of Cas12b in rice by whole genome sequencing (WGS). 1) Major activities competed: Four AaCas12b genome editing systems were closely compared in the rice protoplast system at the two target sites. The genome editing efficiency was quantified by NGS of PCR amplicons that span the target sites. The top two Cas12b editing systems were further assessed in rice stable lines. Transgenic lines were genotyped by the Hi-Tom NGS platform. Representative T0 lines for Cas12b-Aac and Cas12b-Aa1.2 with simultaneous editing at OsEPFL9 and OsGS3 target genes and different controls were subjected to whole-genome sequencing (WGS). T1 population plants from a few select T0 parental lines were analyzed for Mendelian segregation of the targeted edits and CRISPR-Cas12b transgenes. 2) Data collected: NGS data were collected for PCR amplicons corresponding to three biological replicates of the protoplast assays for the four Cas12b editing systems at two independent target sites. PCR amplicon-based Hi-Tom data of T1 individual plants were collected for genotyping and inheritance analysis. 3) Summary statistics and discussion of results: The rice protoplast data suggest that AaCas12b with Aac-sgRNA scaffold and Aa-sgRNA1.2 scaffold showed high editing efficiency. The WGS data from 13 T0 rice lines revealed undetectable sgRNA-dependent and independent off-target mutations in the rice genome. Further Mendelian segregation analysis suggests Cas12b-edited rice plants with the CRISPR transgenes were readily identified in the next generation. 4) Key outcomes or other accomplishments realized: Two efficient Cas12b editing systems were identified. WGS analysis of stable Cas12b-edited and control lines showed that AaCas12b is a highly specific genome editing system in rice and likely in other plants as well. All the data have been recently published in a paper entitled "On and off-target analyses of CRISPR-Cas12b genome editing systems in rice", which is the cover paper of The CRISPR Journal. This marks the completion of Objective 1 in this project. Objective 2: Assess off-target effects of a highly efficient multiplex Cas12a system in rice by WGS. 1) Major activities competed: Three multiplexed CRISPR-Cas12a constructs were made, including one multiplexed LbCas12a construct for simultaneous editing of four target sites, one multiplexed Mb2Cas12a construct for simultaneous editing of four target sites, and one Multiplexed LbCas12a construct for simultaneous editing of 16 target sites. These constructs were used for rice transformation. The resulting transgenic lines were genotyped by NGS using barcoded primers to reveal mutations at each target site. Selected edited T0 rice lines from the three constructs, along with control lines, were subjected to WGS using a illumine HiSeq platform. 2) Data collected: About 15 to 21 independent T0 lines were genotyped for each of the three multiplexed Cas12a constructs. WGS data were collected for a total of 16 plants, including different controls. 3) Summary statistics and discussion of results: Multiplexed Cas12a genome editing efficiency were scored. When multiplexing four target sites, LbCas12a and Mb2Cas12a resulted in nearly 100% editing efficiency with over 80% biallelic editing efficiency. When multiplexing 16 target sites, 20 out of 21 T0 lines contained edits at 13 or more sites with seven or more biallelic edits. Eleven T0 lines had edits at 15 sites, and one line had all 16 sites edited. The average editing and biallelic editing efficiencies for each target were 89.3% and 69.8%, respectively, indicating high multiplexed editing frequency. 4) Key outcomes or other accomplishments realized: To reveal any structural variations due to multiplexed editing, we used manta, lumpy, and grids software programs to analyze the WGS data. We only detected two chromosomal structural changes involving translocations between rice chromosome 3 and 7 due to simultaneous DNA double-strand breaks. These events were further validated by PCR and Sanger sequencing. Overall, it appears to be rare to have such chromosomal structural changes due to multiplexed genome editing. These data have been published in a paper entitled "Genome-wide investigation of multiplexed CRISPR-Cas12a mediated editing in rice" in The Plant Genome. This marks the completion of Objective 2 in this project. Objective 3: Assess off-target effects of prime editors in rice at the genome and transcriptome levels. 1) Major activities competed: We tested prime editing in rice with two platforms. The first platform includes PE2, PE3, PE4 and PE5. A total of 21 T-DNA constructs were made to test these Pes at two endogenous target sites in the rice genome including no-sgRNA negative controls. The second platform includes CRISPR-Combo based PE4 and PE5 systems. Six T-DNA constructs were made to conduct multiplexed prime editing at up to 4 target sites at once. These constructs were used for rice transformation through the Agrobacterium-mediated transformation of Kitaake. The resulting transgenic lines were genotyped in pooled samples by NGS using barcoded Hi-Tom primers with the Illumina HiSeq platform to reveal mutations at each target site. 2) Data collected: About 50 independent T0 lines were genotyped for each of the prime editing constructs. So far, 350 T0 lines have been generated and genotyped by NGS of PCR amplicons. 3) Summary statistics and discussion of results: No positive prime editing events have been identified among the T0 lines genotyped for the PE2 to PE5 system of the first platform, suggesting that prime editing efficiency is very low or there were some problems with these vectors. However, we obtained up to 20% PE efficiency with some of our CRISPR-Combo PE4 (PEA4) and PE5 (PEA5) systems, which will allow us to further improve prime editing using the hormone-free tissue culture method that we recently established in rice. 4) Key outcomes or other accomplishments realized: PEA4 and PEA5 generated comparable prime editing efficiencies to PE4 and PE5, respectively, in rice protoplasts. Based on the protoplast assay, PEA5 and PE5 are slightly more efficient than PEA4 and PE4. However, in stable transgenic rice, PEA4 and PEA5 generated similar editing efficiency. We are currently focusing on further improvement of the PE systems in rice based on the CRISPR-Combo platform.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Changtian Pan, Yiping Qi. Targeted Activation of Arabidopsis Genes by a Potent CRISPR-Act3.0 System. Methods Mol Biol. 2023:2698:27-40. doi: 10.1007/978-1-0716-3354-0_3.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Aytug Tuncel, Changtian Pan, Thorben Sprink, Ralf Wilhelm, Rodolphe Barrangou, Li Li, Patrick M. Shih, Rajeev K. Varshney, Leena Tripathi, Joyce Van Eck, Kranthi Mandadi & Yiping Qi. Nature Reviews Bioengineering volume 1, pages 799816 (2023). https://www.nature.com/articles/s44222-023-00115-8
• Type: Journal Articles Status: Published Year Published: 2024 Citation: Xu Tang, Ayman Eid, Rui Zhang, Yanhao Cheng, Annan Liu, Yurong Chen, Pengxu Chen, Yong Zhang, Yiping Qi. Genome editing in rice and tomato with a small Un1Cas12f1 nuclease. The Plant Genome. 28 May 2024. https://doi.org/10.1002/tpg2.20465

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:This project will provide cutting-edge training on plant genome editing technology development and evaluation for a post-doc, as well as multiple undergraduate and high school interns. The new CRISPR genome editing systems with improved editing activity will directly benefit many plant scientists who are interested in applying this exciting technology for foundational and applied research. The off-targeting data resulting from whole genome sequencing and RNA sequencing will guide breeders in the public sector and private industry in choosing appropriate CRISPR technologies for achieving their product development goals. The off-target information on Cas12b, multiplex Cas12a and prime editing will also aid government agencies, such as USDA and FDA, to make science-based regulatory rules and rational decisions. In this reporting period, the PI has presented the work and acknowledged this NIFA funding in the following presentations: 1. August 2, 2023. New Frontiers-Cell To Seed: Revolutions in Breeding Technologies, Corteva Agriscience, Johnston, IA. 2. July 13, 2023. Gates Masterclass on construct design for crop engineering, gene regulation and genome editing. Online. 3. March 17, 2023. Department of Genetics and Biochemistry. Clemson University, Clemson, SC. 4. February 14, 2023. The 5th Annual CRISPR AgBio Congress, San Diego, CA. 5. February 7, 2023. Center for Computational & Integrative Biology, Rutgers-Camden, New Jersey. 6. January 19, 2023. International Seminar and Workshop on CRISPR/Cas-based Plant Functional Genomics and Computational Modeling, Jan 18-21, 2023. Jorhat, Assam, India. 7. January 15, 2023. Plant and Animal Genome Conference, Jan 13-18, 2023. San Diego, CA. 8. January 13, 2023. Plant and Animal Genome Conference, Jan 13-18, 2023. San Diego, CA. 9. November 18, 2022. The 2nd International Symposium for Horticultural Plant Biology and Biotechnology. Beijing, China. 10. October 31, 2022. The 5th International Conference on CRISPR Technologies. Berkeley, CA. 11. October 24, 2022. The 9th Plant Genomics and Gene Editing Congress (USA). Raleigh, NC. 12. September 14, 2022. The Center of Soybean Research of the Chinese University of Hong Kong & USDA's Agricultural Trade Office (ATO) in Hong Kong. The audience of these presentations was very diverse, including students, academic researchers, industry researchers and governmental officials from different countries. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training opportunities for three postdocs, one PhD student, one visiting PhD student, and one undergraduate intern in the Qi lab. How have the results been disseminated to communities of interest?The WGS data for off-target analysis of multiplexed Cas12a systems have been deposited to the NCBI public database. The WGS data for on and off-target analysis of multiple Cas12b systems have also been deposited to NCBI. Two manuscripts, corresponding to Objective 1 and Objective 2, have been published in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?To date, most of the experiments have been done according to our timeline. Importantly, Objectives 1 and 2 have been finished and published. Due to the COVID-19 pandemic and personnel changes, we delayed our start time for Objective 3 on developing prime editors and analyzing the off-target effects of these prime editors. Also, we met some technical challenges in identifying positive rice lines that were edited by prime editing, with the PE systems in the first platform that we used. However, we have recently solved the problem and our CRISPR-Combo-based PE systems are very promising. In the next reporting period, we will focus on these vectors and generate prime edited lines for further GWS analysis of any potential off-target effects of these new PE systems.

Impacts
What was accomplished under these goals? CRISPR-Cas12b, multiplexed Cas12a, and Cas9-based prime editors are exciting genome editing tools that help create precise mutations in elite cultivars, hence greatly accelerating crop breeding. However, there is a key knowledge gap regarding any potential off-target effects of these three emerging new genome editing technologies. This knowledge gap can be filled by a comprehensive analysis of the edited plants through whole genome sequencing (WGS). In this project, we will use WGS to assess the potential off-target effects of these genome editing systems in rice, a major stable crop feeding nearly half of the world population. Importantly, we will assess the off-target effects of these new tools in precisely edited rice plants by WGS and by including all necessary control plants. Impact: These three new CRISPR systems have great promise in the precision breeding of resilient crops that can fight global hunger and catch up with climate change. The off-target information of these genome editing tools resulting from this project will aid regulatory agencies worldwide to guide the proper use of these new breeding technologies in a science-based framework. We specifically address the BRAG program area "5g", where it calls for a comparison of the types and frequencies of nucleic acid changes introduced into important crop genomes, via genetic engineering techniques versus other plant breeding techniques. Objective 1. Assess off-target effects of Cas12b in rice by whole genome sequencing (WGS). 1) Major activities competed: Four AaCas12b genome editing systems were closely compared in the rice protoplast system at the two target sites. The genome editing efficiency was quantified by NGS of PCR amplicons that span the target sites. The top two Cas12b editing systems were further assessed in rice stable lines. Transgenic lines were genotyped by the Hi-Tom NGS platform. Representative T0 lines for Cas12b-Aac and Cas12b-Aa1.2 with simultaneous editing at OsEPFL9 and OsGS3 target genes and different controls were subjected to whole-genome sequencing (WGS). T1 population plants from a few select T0 parental lines were analyzed for Mendelian segregation of the targeted edits and CRISPR-Cas12b transgenes. 2) Data collected: NGS data were collected for PCR amplicons corresponding to three biological replicates of the protoplast assays for the four Cas12b editing systems at two independent target sites. PCR amplicon-based Hi-Tom data of T1 individual plants were collected for genotyping and inheritance analysis. 3) Summary statistics and discussion of results: The rice protoplast data suggest that AaCas12b with Aac-sgRNA scaffold and Aa-sgRNA1.2 scaffold showed high editing efficiency. The WGS data from 13 T0 rice lines revealed undetectable sgRNA-dependent and independent off-target mutations in the rice genome. Further Mendelian segregation analysis suggests Cas12b-edited rice plants with the CRISPR transgenes were readily identified in the next generation. 4) Key outcomes or other accomplishments realized: Two efficient Cas12b editing systems were identified. WGS analysis of stable Cas12b-edited and control lines showed that AaCas12b is a highly specific genome editing system in rice and likely in other plants as well. All the data have been recently published in a paper entitled "On and off-target analyses of CRISPR-Cas12b genome editing systems in rice", which is the cover paper of The CRISPR Journal. This marks the completion of Objective 1 in this project. Objective 2: Assess off-target effects of a highly efficient multiplex Cas12a system in rice by WGS. 1) Major activities competed: Three multiplexed CRISPR-Cas12a constructs were made, including one multiplexed LbCas12a construct for simultaneous editing of four target sites, one multiplexed Mb2Cas12a construct for simultaneous editing of four target sites, and one Multiplexed LbCas12a construct for simultaneous editing of 16 target sites. These constructs were used for rice transformation. The resulting transgenic lines were genotyped by NGS using barcoded primers to reveal mutations at each target site. Selected edited T0 rice lines from the three constructs, along with control lines, were subjected to WGS using a illumine HiSeq platform. 2) Data collected: About 15 to 21 independent T0 lines were genotyped for each of the three multiplexed Cas12a constructs. WGS data were collected for a total of 16 plants, including different controls. 3) Summary statistics and discussion of results: Multiplexed Cas12a genome editing efficiency were scored. When multiplexing four target sites, LbCas12a and Mb2Cas12a resulted in nearly 100% editing efficiency with over 80% biallelic editing efficiency. When multiplexing 16 target sites, 20 out of 21 T0 lines contained edits at 13 or more sites with seven or more biallelic edits. Eleven T0 lines had edits at 15 sites, and one line had all 16 sites edited. The average editing and biallelic editing efficiencies for each target were 89.3% and 69.8%, respectively, indicating high multiplexed editing frequency. 4) Key outcomes or other accomplishments realized: To reveal any structural variations due to multiplexed editing, we used manta, lumpy, and grids software programs to analyze the WGS data. We only detected two chromosomal structural changes involving translocations between rice chromosome 3 and 7 due to simultaneous DNA double-strand breaks. These events were further validated by PCR and Sanger sequencing. Overall, it appears to be rare to have such chromosomal structural changes due to multiplexed genome editing. These data have been published in a paper entitled "Genome-wide investigation of multiplexed CRISPR-Cas12a mediated editing in rice" in The Plant Genome. This marks the completion of Objective 2 in this project. Objective 3: Assess off-target effects of prime editors in rice at the genome and transcriptome levels. 1) Major activities competed: We tested prime editing in rice with two platforms. The first platform includes PE2, PE3, PE4 and PE5. A total of 21 T-DNA constructs were made to test these Pes at two endogenous target sites in the rice genome including no-sgRNA negative controls. The second platform includes CRISPR-Combo based PE4 and PE5 systems. Six T-DNA constructs were made to conduct multiplexed prime editing at up to 4 target sites at once. These constructs were used for rice transformation through the Agrobacterium-mediated transformation of Kitaake. The resulting transgenic lines were genotyped in pooled samples by NGS using barcoded Hi-Tom primers with the Illumina HiSeq platform to reveal mutations at each target site. 2) Data collected: About 50 independent T0 lines were genotyped for each of the prime editing constructs. So far, 350 T0 lines have been generated and genotyped by NGS of PCR amplicons. 3) Summary statistics and discussion of results: No positive prime editing events have been identified among the T0 lines genotyped for the PE2 to PE5 system of the first platform, suggesting that prime editing efficiency is very low or there were some problems with these vectors. However, we obtained up to 20% PE efficiency with some of our CRISPR-Combo PE4 (PEA4) and PE5 (PEA5) systems, which will allow us to further improve prime editing using the hormone-free tissue culture method that we recently established in rice. 4) Key outcomes or other accomplishments realized: PEA4 and PEA5 generated comparable prime editing efficiencies to PE4 and PE5, respectively, in rice protoplasts. Based on the protoplast assay, PEA5 and PE5 are slightly more efficient than PEA4 and PE4. However, in stable transgenic rice, PEA4 and PEA5 generated similar editing efficiency. We are currently focusing on PEA4 to demonstrate CRISPR-Combo based PE systems in rice. The resulting plants will be analyzed by WGS.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Changtian Pan, Yiping Qi. PrimeRoot for targeted large DNA insertion in plants. Trends in Plant Science. May 24, 2023. https://doi.org/10.1016/j.tplants.2023.05.002
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Liyang Zhang, Gen Li, Yingxiao Zhang, Yanhao Cheng, Steve Glenn, Michael Collingwood, Nicole Bode, Sarah Beaudoin, Christopher Vakulskas*, Yiping Qi. Boosting genome editing efficiency in human cells and plants with novel Lbcas12a variants. Genome Biology. 2023. 24, Article number: 102. https://genomebiology.biomedcentral.com/articles/10.1186/s13059-023-02929-6
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Changtian Pan, Yiping Qi. CRISPR-Combomediated orthogonal genome editing and transcriptional activation for plant breeding. Nature Protocols. 2023. https://www.nature.com/articles/s41596-023-00823-w
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Changtian Pan, Gen Li, Anindya Bandyopadhyay, Yiping Qi*. Guide RNA library-based CRISPR screens in plants: opportunities and challenges. Current Opinion in Biotechnology, 2023, 79: 102883. https://doi.org/10.1016/j.copbio.2022.102883
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Filiz Gurel, Yuechao Wu, Changtian Pan, Gen Li, Tao Zhang, Yiping Qi. On-and Off-Target Analyses of CRISPR-Cas12b Genome Editing Systems in Rice. The CRISPR Journal. 2023. https://doi.org/10.1089/crispr.2022.0072
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Yingxiao Zhang, Yuechao Wu, Gen Li, Aileen Qi, Yong Zhang, Tao Zhang, Yiping Qi. Genome-wide investigation of multiplexed CRISPR-Cas12a-mediated editing in rice. The Plant Genome. 2023. https://doi.org/10.1002/tpg2.20266
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Aytug Tuncel, Yiping Qi. CRISPR/Cas mediated genome editing in potato: Past achievements and future directions. Plant Science. 2022. https://doi.org/10.1016/j.plantsci.2022.111474
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Naga Rajitha Kavuri, Manikandan Ramasamy, Yiping Qi, Kranthi Mandadi. Applications of CRISPR/Cas13-Based RNA Editing in Plants. Cells. 2022. 11 (17), 2665. https://www.mdpi.com/2073-4409/11/17/2665

Progress 09/01/21 to 08/31/22

Outputs
Target Audience:This project will provide cutting-edge training on plant genome editing technology development and evaluation for a post-doc, as well as multiple undergraduate and high school interns. The new CRISPR genome editing systems with improved editing activity will directly benefit many plant scientists who are interested in applying this exciting technology for foundational and applied research. The off-targeting data resulting from whole genome sequencing and RNA sequencing will guide breeders in the public sector and private industry in choosing appropriate CRISPR technologies for achieving their product development goals. The off-target information on Cas12b, multiplex Cas12a and prime editing will also aid government agencies, such as USDA and FDA, to make science-based regulatory rules and rational decisions. In this reporting period, the PI has presented the work and acknowledged this NIFA funding in the following presentations: 1. August 10, 2022. The 3rd International Symposium of Horticulture and Plant Biology, Huazhong Agriculture University, Wuhan. 2. July 29, 2022. 2022 International Symposium on Plant Biotic Interactions and Plant Health, Wuhan, China. 3. July 10, 2022. Plant Biology 2022. ASPB and CSPB/SCBV. Portland, USA. 4. July 7, 2022. The 20th IUFRO Tree Biotech Meeting, Harbin, China. 5. June 29, 2022. PlantEd COST Action - plant genome editing. European Cooperation in Science and Technology. 6. June 7, 2022. 2022 In Vitro Biology Meeting, San Diego, CA. 7. June 5, 2022. 2022 In Vitro Biology Meeting, San Diego, CA. 8. May 29, 2022. The 23rd Penn State Symposium in Plant Biology -- RNA Biology. State College, PA. 9. May 25, 2022. Novel Crop Breeding Techniques: Towards more sustainable agriculture. 10. February 10, 2022. Dale Bumpers National Rice Research Center-USDA-ARS. Stuttgart, AR. 11. January 10, 2022. Plant and Animal Genome XXIX Conference, Jan 8-12, 2022. San Diego, CA. 12. 2021 ASA, CSSA,SSSAINTERNATIONALANNUAL MEETING NOVEMBER 7-10, SALTLAKE CITY, UT. Symposium--Frontiers in Plant Genome Editing for Crop Improvement. 13. September 15, 2021. 2nd Congress of Geneticists in Bosnia and Herzegovina with International Participation. 14. September 14, 2021. Virtual Workshop on Advanced Biotechnology in Bosnia and Herzegovina. 15. September 2, 2021. National Center for Genome Editing in Agriculture, Israel. 16. August 17, 2021. CTC Genomics, St. Louis, Missouri. The audiences of these presentations were very diverse, including students, academic researchers, industry researchers, and government officials from different countries. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training opportunities for one postdoc and one undergraduate intern in the Qi lab. How have the results been disseminated to communities of interest?The WGS data for off-target analysis of multiplexed Cas12a systems have been deposited into NCBI public database. The WGS data for on and off-target analysis of multiple Cas12b systems have also been deposited to NCBI. Two manuscripts, corresponding to Objective 1 and Objective 2, have been submitted to peer-reviewed journals for publication. What do you plan to do during the next reporting period to accomplish the goals?To date, most of the experiments have been done according to our timeline. Importantly, Objectives 1 and 2 have been finished. Due to the Covid-19 pandemic and personnel changes, we delayed our start time for Objective 3 on developing prime editors and analyzing of off-target effects of these prime editors. Although we have started our work in Objective 3 in this reporting period, there is a significant challenge to developing a reliable and robust prime editor in plants. In the next reporting period, we will focus on research activities in Objective 3 by trying more innovative ideas. We hope to identify and assess a much-improved plant prime editor, which is likely based on our innovative CRISPR-Combo platform.

Impacts
What was accomplished under these goals? CRISPR-Cas12b, multiplexed Cas12a, and Cas9-based prime editors are exciting genome editing tools that help create precise mutations in elite cultivars, hence greatly accelerating crop breeding. However, there is a key knowledge gap regarding any potential off-target effects of these three emerging new genomes editing technologies. This knowledge gap can be filled by a comprehensive analysis of the edited plants through whole genome sequencing (WGS). In this project, we will use WGS to assess potential off-target effects of these genome editing systems in rice, a major stable crop feeding nearly half of the world population. Importantly, we will assess the off-target effects of these new tools in precisely edited rice plants by WGS and by including all necessary control plants. Impact: These three new CRISPR systems have great promise in the precision breeding of resilient crops that can fight global hunger and catch up with climate change. The off-target information of these genome editing tools resulting from this project will aid regulatory agencies worldwide to guide the proper use of these new breeding technologies in a science-based framework. We specifically address the BRAG program area "5g", where it calls for a comparison of the types and frequencies of nucleic acid changes introduced into important crop genomes, via genetic engineering techniques versus other plant breeding techniques. Objective 1. Assess off-target effects of Cas12b in rice by whole genome sequencing (WGS). 1) Major activities competed: Four AaCas12b genome editing systems were closely compared in the rice protoplast system at the two target sites.The genome editing efficiency was quantified by NGS of PCR amplicons that span the target sites. The top two Cas12b editing systems were further assessed in rice stable lines. Transgenic lines were genotyped by the Hi-Tom NGS platform. Representative T0 lines for Cas12b-Aac and Cas12b-Aa1.2 with simultaneous editing at OsEPFL9 and OsGS3 target genes and different controls were subjected to whole-genome sequencing (WGS). T1 population plants from a few select T0 parental lines were analyzed for Mendelian segregation of the targeted edits and CRISPR-Cas12b transgenes. 2) Data collected: NGS data were collected for PCR amplicons corresponding to three biological replicates of the protoplast assays for the four Cas12b editing systems at two independent target sites. PCR amplicon-based Hi-Tom data of T1 individual plants were collected for genotyping and inheritance analysis. 3) Summary statistics and discussion of results: The rice protoplast data suggest that AaCas12b with Aac-sgRNA scaffold and Aa-sgRNA1.2 scaffold showed highediting efficiency. The WGS data from 13 T0 rice lines revealed undetectable sgRNA-dependent and independent off-target mutations in the rice genome. Further Mendelian segregation analysis suggests Cas12b-edited rice plants with the CRISPR transgenes were readily identified in the next generation. 4) Key outcomes or other accomplishments realized: Two efficient Cas12b editing systems were identified. WGS analysis of stable Cas12b-edited and control lines showed that AaCas12b is a highly specific genome editing system in rice and likely in other plants as well. All the data have been organized into a manuscript entitled "On and off-target analyses of CRISPR-Cas12b genome editing systems in rice" which is currently under review in a peer-reviewed journal. This marks the completion of Objective 1 in this project. Objective 2: Assess off-target effects of a highly efficient multiplex Cas12a system in rice by WGS. 1) Major activities competed: Three multiplexed CRISPR-Cas12a constructs were made, including one multiplexed LbCas12a construct for simultaneous editing of four target sites, one multiplexed Mb2Cas12a construct for simultaneous editing of four target sites, and one Multiplexed LbCas12a construct for simultaneous editing of 16 target sites. These constructs were used for rice transformation. The resulting transgenic lines were genotyped by NGS using barcoded primers to reveal mutations at each target site. Selected edited T0 rice lines from the three constructs, along with control lines, were subjected to WGS using a illumine HiSeq platform. 2) Data collected: About 15 to 21 independent T0 lines were genotyped for each of the three multiplexed Cas12a constructs. WGS data were collected for a total of 16 plants, including different controls. 3) Summary statistics and discussion of results: Multiplexed Cas12a genome editing efficiency were scored. When multiplexing four target sites, LbCas12a and Mb2Cas12a resulted in nearly 100% editing efficiency with over 80% biallelic editing efficiency. When multiplexing 16 target sites, 20 out of 21 T0 lines contained edits at 13 or more sites with seven or more biallelic edits. Eleven T0 lines had edits at 15 sites, and one line had all 16 sites edited. The average editing and biallelic editing efficiencies for each target were 89.3% and 69.8%, respectively, indicating high multiplexed editing frequency. 4) Key outcomes or other accomplishments realized: To reveal any structural variations due to multiplexed editing, we used manta, lumpy, and grids software programs to analyze the WGS data. We only detected two chromosomal structural changes involving translocations between rice chromosome 3 and 7 due to simultaneous DNA double-strand breaks. These events were further validated by PCR and Sanger sequencing. Overall, it appears to be rare to have such chromosomal structural changes due to multiplexed genome editing. These data have been organized and written in a manuscript entitled "Genome-wide investigation of multiplexed CRISPR-Cas12a mediated editing in rice" which is currently under review in a peer-reviewed journal. This marks the completion of Objective 2 in this project. Objective 3: Assess off-target effects of prime editors in rice at the genome and transcriptome levels. 1) Major activities competed: Gateway Cas9-PE entry clones and the corresponding modified sgRNA entry clones for seven different prime editing strategies were designed and constructed. With these new prime editing systems, 21 T-DNA constructs were made to test at two endogenous target sites in the rice genome including no-sgRNA negative controls. These constructs were used for rice transformation through the Agrobacterium-mediated transformation of Kitaake. The resulting transgenic lines were genotyped in pooled samples by NGS using barcoded Hi-Tom primers with the Illumina HiSeq platform to reveal mutations at each target site. 2) Data collected: About 50 independent T0 lines were genotyped for each of the prime editing constructs. So far, 200 T0 lines have been generated and genotyped by NGS of PCR amplicons. 3) Summary statistics and discussion of results: No positive prime editing events have been identified among the T0 lines genotyped, suggesting that prime editing efficiency is very low, which is consistent with our previous published observation and other literature. 4) Key outcomes or other accomplishments realized: For the four presumably improved prime editors tested in rice, editing efficiency in rice stable lines is too low to be detected within a sizable population (~50 T0 lines per construct). So, the estimated prime editing frequency by these PEs at the two target sites is below 2%. This outcome reinforces the importance to seek for future improvement of prime editing in plants. In the coming year, we will continue to test the remaining three prime editing strategies using this established rice stable plant pipeline. Meanwhile, we will also start exploring the use of CRISPR-Combo (an innovative technology developed in the PI's lab). We expect to see promising results with PEs developed based on the CRISPR-Combo platform.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Changtian Pan, Gen Li, Aimee A. Malzahn, Benjamin Leyson, Simon Sretenovic, Yanhao Cheng, Filiz Gurel, Gary D. Coleman, Yiping Qi. Boosting plant genome editing with a versatile CRISPR-Combo system. Nature Plants, 2022. https://www.nature.com/articles/s41477-022-01151-9
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Ayman Eid, Yiping Qi. Prime editor integrase systems boost targeted DNA insertion and beyond. Trends in Biotechnology. 2022, 40 (8): 907-909. https://doi.org/10.1016/j.tibtech.2022.05.002
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Yanhao Cheng, Simon Sretenovic, Yingxiao Zhang, Ji Huang, Yiping Qi. Expanding the targeting scope of FokI-dCas nuclease systems with SpRY and Mb2Cas12a. Biotechnology Journal. 2022 Apr 4;e2100571. https://onlinelibrary.wiley.com/doi/10.1002/biot.202100571
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Shaun Curtin, Yiping Qi, L�zaro EP Peres, Alisdair R Fernie, Agustin Zs�g�n. Pathways to de novo domestication of crop wild relatives. Plant Physiology, 2021, 188 (4): 1746-1756. https://doi.org/10.1093/plphys/kiab554
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Yingxiao Zhang, Yanhao Cheng, Hong Fang, Nathaniel Roberts, Liyang Zhang, Christopher A. Vakulskas, Randall P. Niedz, James N. Culver, Yiping Qi. Highly Efficient Genome Editing in Plant Protoplasts by Ribonucleoprotein Delivery of CRISPR-Cas12a Nucleases. Frontiers in Genome Editing, 2022. https://doi.org/10.3389/fgeed.2022.780238
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Md. Mahmudul Hassan, Yingxiao Zhang, Guoliang Yuan, Kuntal De, Jin-Gui Chen, Wellington Muchero, Gerald A. Tuskan, Yiping Qi, Xiaohan Yang*. Construct design for CRISPR/Cas-based genome editing in plants. Trends in Plant Science, 2021. https://www.cell.com/trends/plant-science/fulltext/S1360-1385(21)00157-6
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Rajeev K Varshney, Rutwik Barmukh, Manish Roorkiwal, Yiping Qi, Jana Kholova, Roberto Tuberosa, Matthew P Reynolds, Francois Tardieu, Kadambot HM Siddique. Breeding custom-designed crops to improve drought adaptation. Advanced Genetics, 2021. https://onlinelibrary.wiley.com/doi/pdf/10.1002/ggn2.10052

Progress 09/01/20 to 08/31/21

Outputs
Target Audience:This project will provide cutting-edge training on plant genome editing technology development and evaluation for a post-doc, as well as multiple undergraduate and high school interns. The new CRISPR genome editing systems with improved editing activity will directly benefit many plant scientists who are interested in applying this exciting technology for foundational and applied research. The off-targeting data resulted from whole genome sequencing and RNA sequencing will guide breeders in the public sector and private industry in choosing appropriate CRISPR technologies for achieving their product development goals. The off-target information on Cas12b, multiplex Cas12a and prime editing will also aid government agencies, such as USDA and FDA, to make science-based regulatory rules and rational decisions. In this reporting period, the PI has presented the work and acknowledged this NIFA funding in the following presentations: 1. July 28, 2021. Genome Writers Guild (GWG) Virtual Conference. University of Minnesota. 2. July 21, 2021. Reliance Industries Limited (RIL) R&D Community Webinar Series. 3. June 6, 2021. SIVB 2021: In Vitro OnLine. Session: Variables Controlling Successful Gene Editing in Plants/New Tools. 4. March 31, 2021. Genome Editing Webinar series organized by BioDesign Research and Nanjing Agriculture University in China. 5. March 25, 2021. Hands-on Laboratory Course on CRISPR-Cas Gene Editing. SGT University, Gurgaon & Alliance of Bioversity International and CIAT, Asia-India, New Delhi, India. [Virtual presentation]. 6. March 9, 2021. Keystone eSymposia- Plant Genome Engineering: From Lab to Field. 7. March 3, 2021. 8th Plant Genomes & Gene Editing Congress: USA. Virtual Conference. 8. February 25, 2021. Department of Biology, East Carolina University, Greenville, NC. 9. January 19, 2021. Cellfies Symposium, Hosted by Bayer AgConnect. 10. October 20, 2020. 2nd International Plant Genetics and Genomics Symposium-IPGG, Assiut University, Egypt. 11. September 17, 2020. Department of Biology, University of Pennsylvania, PA. 12. September 14, 2020. International workshop on plant genome editing, Paris, France. The audience of these presentations was very diverse, including students, academic researchers, industry researchers and governmental officials from different countries. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training opportunities for one postdoc and one high school intern in the Qi lab. How have the results been disseminated to communities of interest?The WGS data for off-target analysis of multiplexed Cas12a systems have been deposited to NCBI public database. What do you plan to do during the next reporting period to accomplish the goals?To date, most of the experiments have been done according to our timeline. Due to the Covid-19 pandemic and personnel changes, we delayed our start time for Objective 3 on prime editors. In the next reporting period, we plan to finish Objective 2, largely finish Objective 1, and start research activities in Objective 3.

Impacts
What was accomplished under these goals? CRISPR-Cas12b, multiplexed Cas12a, and Cas9-based prime editors are exciting genome editing tools that help create precise mutations in elite cultivars, hence greatly accelerating crop breeding. However, there is a key knowledge gap regarding any potential off-target effects by these three emerging new genomes editing technologies. This knowledge gap can be filled by comprehensive analysis of the edited plants through whole genome sequencing (WGS). In this project, we will use WGS to assess potential off-target effects of these genome editing systems in rice, a major stable crop feeding nearly half of the world population. Importantly, we will assess off-target effects of these new tools in precisely edited rice plants by WGS and by including all necessary control plants. Impact: These three new CRISPR systems have great promise in precision breeding of resilient crops that can fight global hunger and catch up with climate change. The off-target information of these genome editing tools resulting from this project will aid regulatory agencies worldwide to guide proper use of these new breeding technologies in a science-based framework. We specifically address the BRAG program area "5g", where it calls for comparison of the types and frequencies of nucleic acid changes introduced into important crop genomes, via genetic engineering techniques versus other plant breeding techniques. Objective 1. Assess off-target effects of Cas12b in rice by whole genome sequencing (WGS).1) Major activities competed: Four AaCas12b genome editing systems were closely compared in the rice protoplast system, which are AaCas12b with Aac-sgRNA scaffold, AaCas12b with Aa-sgRNA1.2 scaffold, AaCas12b-TV with Aa3.8 scaffold, and AaCas12b-TV-MS2-VPR with Aac.3 scaffold. These four Cas12b systems were tested at the two target sites: OsEPFL9-sgRNA02 and OsGS3-sgRNA02. The genome editing efficiency was quantified by NGS of PCR amplicons that span the target sites. Top two Cas12b editing systems were further assessed in rice stable transformants resulting from Agrobacterium mediated transformation. Transgenic lines were genotyped by the Hi-Tom NGS platform. 2) Data collected: NGS data were collected for PCR amplicons corresponding to three biological replicates of the protoplast assays for the four Cas12b editing systems at two independent targets sites. We are currently collecting additional NGS data for genotyping individual T0 rice lines for genome editing at the two target sites with the Hi-Tom platform. Quantitative RT-PCR was used for screening transgenic GFP controls lines and three high expression lines were identified, to be used for WGS. 3) Summary statistics and discussion of results: The rice protoplast data suggest that AaCas12b with Aac-sgRNA scaffold and Aa-sgRNA1.2 scaffold showed higher editing efficiency than the other two Cas12b systems. These two top Cas12b systems were used for generating stable rice lines for further comparison of genome editing efficiency in multiplexed editing manner. The results will help us identify edited for WGS to reveal any off-target effects. In addition, transgenic plants that express Cas12b only and GFP were also made and will be used as controls for WGS. 4) Key outcomes or other accomplishments realized: Two efficient Cas12b editing systems were identified and their resulting edited lines will be subjected for WGS soon. We expect to wrap up the WGS and analysis within the next reporting period. Objective 2: Assess off-target effects of a highly efficient multiplex Cas12a system in rice by WGS. 1) Major activities competed: Three multiplexed CRISPR-Cas12a constructs were made, including one multiplexed LbCas12a construct for simultaneous editing of four target sites, one multiplexed Mb2Cas12a construct for simultaneous editing of four target sites, and one Multiplexed LbCas12a construct for simultaneous editing of 16 target sites. These constructs were used for rice transformation through Agrobacterium mediated transformation of Kitaake, a Japonica rice variety. The resulting transgenic lines were genotyped by NGS using barcoded primers (with the Hi-Tom platform) to reveal mutations at each target site. Selected edited T0 rice lines from the three constructs, along with control lines, were subjected to WGS using a illumine HiSeq platform. 2) Data collected: About 15 to 21 independent T0 lines were genotyped for each of the three multiplexed Cas12a constructs. WGS data were collected for a total of 16 plants, including three wild type plants (non-tissue cultured plants), two tissue cultured plants, two LbCas12a expression lines without a guide RNA, two Mb2Cas12a expression lines without a guide RNA, two multiplexed editing lines by LbCas12a, two multiplexed editing lines by Mb2Cas12a, and three multiplexed editing lines by LbCas12a. 3) Summary statistics and discussion of results: Multiplexed Cas12a genome editing efficiency were scored. When multiplexing four target sites, LbCas12a and Mb2Cas12a resulted in nearly 100% editing efficiency with over 80% biallelic editing efficiency. When multiplexing 16 target sites, 20 out of 21 T0 lines contained edits at 13 or more sites with seven or more biallelic edits. Eleven T0 lines had edits at 15 sites, and one line had all 16 sites edited. The average editing and biallelic editing efficiencies for each target were 89.3% and 69.8%, respectively, indicating high multiplexed editing frequency. 4) Key outcomes or other accomplishments realized: To reveal any structural variations due to multiplexed editing, we used manta, lumpy and grids software programs to analyze the WGS data. We only detected two chromosomal structural changes involving translocations between rice chromosome 3 and 7 due to simultaneous DNA double strand breaks. Currently, we are further validating these events by PCR. Overall, it appears to be rare to have such chromosomal structural changes due to multiplexed genome editing. The discovery along with further findings will soon be written as a manuscript for submission to a peer reviewed journal. Objective 3: Assess off-target effects of prime editors in rice at the genome and transcriptome levels. We prioritized our research activities on the first two Objectives in this reporting period. The research on prime editors has been planned and adjusted based on recent publications on this topic. Our progress on Objective 3 will be reported in next reporting period.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Changtian Pan, Xincheng Wu, Kasey Markel, Aimee A. Malzahn, Neil Kundagrami, Simon Sretenovic, Yingxiao Zhang, Yanhao Cheng, Patrick M Shih, Yiping Qi. CRISPR-Act3.0 for highly efficient multiplexed gene activation in plants. Nature Plants, 2021. https://www.nature.com/articles/s41477-021-00953-7
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Qiurong Ren, Simon Sretenovic, Guanqing Liu, Zhaohui Zhong, Jiaheng Wang, Lan Huang, Xu Tang, Yachong Guo, Li Liu, Yuechao Wu, Jie Zhou, Yuxin Zhao, Han Yang, Yao He, Shishi Liu, Desuo Yin, Rocio Mayorga, Xuelian Zheng, Tao Zhang, Yiping Qi, Yong Zhang. Improved plant cytosine base editors with high editing activity, purity, and specificity. Plant Biotechnology Journal, 2021. https://doi.org/10.1111/pbi.13635
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Jianping Zhou, Mingzhu Yuan, Yuxin Zhao, Quan Quan, Dong Yu, Han Yang, Xu Tang, Xuhui Xin, Guangze Cai, Qian Qian, Yiping Qi, Yong Zhang. Efficient deletion of multiple circle RNA loci by CRISPR?Cas9 reveals Os06circ02797 as a putative sponge for OsMIR408 in rice. Plant Biotechnology Journal, 2021, https://doi.org/10.1111/pbi.13544
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Gen Li, Simon Sretenovic, Edward Eisenstein, Gary Coleman, Yiping Qi. Highly efficient C-to-T and A-to-G base editing in a Populus hybrid. Plant Biotechnology Journal, 2021 Mar 20. doi: 10.1111/pbi.13581.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Yingxiao Zhang, Brian Iaffaldano, Yiping Qi. CRISPR ribonucleoprotein-mediated genetic engineering in plants. Plant Communications, 2021, 2 (2): 100168. DOI: 10.1038/s41598-018-32702-w
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Changtian Pan, Simon Sretenovic, Yiping Qi. CRISPR/dCas-mediated transcriptional and epigenetic regulation in plants. Current Opinion in Plant Biology, 2021, 60, 101980. https://doi.org/10.1016/j.pbi.2020.101980
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Qiurong Ren, Simon Sretenovic, Shishi Liu, Xu Tang, Lan Huang, Yao He, Li Liu, Yachong Guo, Zhaohui Zhong, Guanqing Liu, Yanhao Cheng, Xuelian Zheng, Changtian Pan, Desuo Yin, Yingxiao Zhang, Wanfeng Li, Liwang Qi, Chenghao Li, Yiping Qi, Yong Zhang. PAM-less plant genome editing using a CRISPRSpRY toolbox. Nature Plants, 2021, 7: 25-33. doi: 10.1038/s41477-020-00827-4.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Kutubuddin A. Molla, Yiping Qi, Subhasis Karmakar, Mirza J. Baig. Base Editing Landscape Extends to Perform Transversion Mutation. Trends in Genetics, 2020, 36 (12): 899-901. https://doi.org/10.1016/j.tig.2020.09.001