Judul: Design of sgRNA to Edit DLT Gene using CRISPR-Cas9 and In Silico Mutation Prediction in Rice var. Hawara Bunar
Abstrak: ABSTRAK IBNU HALIM. Desain sgRNA untuk Pengeditan Gen DLT dengan CRISPR- Cas9 dan Prediksi Mutasi secara in silico pada Padi varietas Hawara Bunar. Dibimbing oleh MIFTAHUDIN dan MIFTAHUL HUDA FENDIYANTO. Peningkatan hasil padi yang tinggi adalah tujuan utama dalam memenuhi kebutuhan beras yang meningkat seiring meningkatnya jumlah penduduk. Peningkatan hasil padi dapat dicapai dengan memperbaiki karakter morfo-agronomi termasuk tinggi tanaman dan jumlah anakan. Salah satu pendekatan yang dapat digunakan adalah dengan mengedit gen DWARF and LOW TILLER/DLT melalui teknik CRISPR-Cas9. Gen DLT merupakan faktor transkripsi untuk biosintesis Brassinosteroid/BR. Memanipulasi biosintesis ataupun pensinyalan BR sangat mempengaruhi arsitektur padi. Ketepatan penentuan situs gen target pada teknik CRISPR-Cas9 ditentukan oleh urutan 20-nt dalam sgRNA yang didesain melalui program bioinformatik dan wilayah pengikatan yang dikenal sebagai Protospacer Adjacent Motif (PAM). Untuk itu penelitian ini bertujuan mendesain sgRNA dan memprediksi ketepatan mutasi gen DLT pada padi varietas Hawara Bunar. DNA genom padi diisolasi dengan metode Cetyltrimethyl Ammonium Bromide (CTAB). Amplifikasi gen DLT dengan teknik PCR menggunakan satu pasang primer yang didesain dari gen reference. Hasil PCR disekuen dan sekuen partial gen DLT digunakan untuk mendesain sgRNA menggunakan program chopchop.cbu.uib.no. Oligo sgRNA telah berhasil didesain dengan target domain Gras family protein dari gen DLT padi varietas Hawara Bunar. Mutasi diprediksi akan terjadi pada wilayah domain tersebut yaitu posisi nukleotida 1466573 hingga 1466591, dan pada tingkat struktur sekunder protein perubahan yang terjadi akibat mutasi berawal dari asam amino ke 406 hingga ke posisi C terminal. Kata kunci: CRISPR-Cas9, gene editing, DLT, sgRNA, ABSTRACT IBNU HALIM. Design of sgRNA to Edit DLT Gene using CRISPR-Cas9 and In Silico Mutation Prediction in Rice var. Hawara Bunar. Dibimbing oleh MIFTAHUDIN dan MIFTAHUL HUDA FENDIYANTO. Increasing rice yield can be achieved by modifying of plant architecture, including plant height and tiller number. The DWARF AND LOW TILLERRING (DLT) gene is a transcription factor for a gene involved in Brassinosteroid (BR) biosynthesis. Manipulating BR biosynthesis will strongly influence rice architecture. CRISPR-Cas9 is an accurate tool to edit a gene sequence. The accuracy of site determination of CRISPR-Cas9-mediated target gene is determined by the 20-nt sequence in the sgRNA and the binding site known as the Protospacer Adjacent Motif (PAM). For this reason, the study aimed to design sgRNA and predict the accuracy of DLT gene mutation sites in rice var. Hawara Bunar cultivar. Mutation prediction begins with the isolation of rice genomic DNA using the Cetyltrimethyl Ammonium Bromide (CTAB) method, followed by amplification of the exon 1 DLT gene using a primer pair designed from the reference gene. The PCR product was then sequenced, and then use the sequence to design sgRNA. The study has designed sgRNA located on the targeted sequence that corresponding to the Gras family protein domain of the exon1 DLT gene. The mutation sites were predicted to be at the domain site through the parallel alignment of the nucleotide and amino acid sequences of the DLT gene and the reference gene. It is predicted that mutation in nucleotide sequence of the target site corresponding to the protein domain will change the protein structure and its function. Keywords: Bioinformatics, CRISPR-Cas9, DLT,
Keyword: Bioinformatics, CRISPR-Cas9, DLT, sgRNA, Bioinformatics, CRISPR-Cas9, DLT, sgRNA

Judul: sgRNA Design for ROC5 Gene Editing with CRISPR/Cas9 and in silico Mutation Prediction in Rice cv. Hawara Bunar
Abstrak: Produktivitas padi (Oryza sativa L.) perlu ditingkatkan salah satunya dengan ekstensifikasi penanaman pada lahan suboptimal. Hawara Bunar merupakan kultivar padi toleran cekaman kekeringan dan tanah masam yang berpotensi ditanam di lahan suboptimal. Untuk mengoptimalkan hasil panen pada kondisi lahan tersebut, diperlukan pemuliaan untuk menghasilkan padi kultivar Hawara Bunar yang menunjukkan penggulungan daun secara moderat ketika tercekam kekeringan melalui pengeditan gen ROC5 menggunakan sistem CRISPR/Cas9. Langkah awal dalam pengeditan gen ini adalah desain sgRNA dan prediksi mutasi yang akan dihasilkan baik pada sekuen gen maupun protein dari ROC5. Penelitian ini bertujuan mendesain sgRNA dengan efisiensi yang tinggi dan memprediksi mutasi secara in silico dalam pengeditan gen ROC5 pada padi kultivar Hawara Bunar menggunakan sistem CRISPR/Cas9. Penelitian ini dilakukan melalui analisis in silico. Hasil penelitian ini adalah desain sgRNA untuk pengeditan gen ROC5 padi kultivar Hawara Bunar menggunakan sistem CRIPR/Cas9 yaitu 5’-GGAGGCTCTCCCTCGATGCG-3’ dengan PAM 5’-CGG-3’ dan 5’-CTGAGG CGGAGAACGACAGC-3’ dengan PAM 5’-CGG-3’. Kedua sgRNA tersebut masing-masing diprediksi akan memandu mutasi pada ekson ketiga (posisi genomik ke-27493819) dan kedua (posisi genomik ke-27494141) untuk menyebabkan mutasi delesi 5 pb dan insersi 1 pb dan menghasilkan protein ROC5 mutan yang tidak fungsional. Oligodupleks berdasarkan kedua sgRNA tersebut juga telah didesain., The productivity of rice (Oryza sativa L.) needs to be increased. One of efforts is by extensification of rice cultivation on suboptimal land. Hawara Bunar is a rice cultivar that is tolerant to drought stress and acid soil which has the potential to be cultivated on suboptimal land. To optimize yields in such conditions, it is necessary to develop rice cv. Hawara Bunar that show moderate leaf curling when grown under drought stress through editing the ROC5 gene using the CRISPR/Cas9 system. The initial step of this gene editing are the design of sgRNA and prediction of mutations that will be generated in the gene and protein sequences of ROC5 gene. This study aims to design a high efficiency sgRNA for CRISPR/Cas9 based gene editing and predict mutations that will occur in the edited ROC5 gene in rice cv. Hawara Bunar. The research was conducted through in silico analysis. The result of the study was the sgRNA for ROC5 gene editing in rice cv. Hawara Bunar using the CRIPR/Cas9 system has been developed with the sequence of 5'-GGAGGCT CTCCCTCGATGCG-3' with PAM 5'-CGG-3' and 5'-CTGAGGCGGAGAACG ACAGC-3' with PAM 5'-CGG-3'. These two sgRNAs were predicted to guide mutations in the third (at 27493819 genomic position) and second (at 27494141 genomic position) exons of the ROC5 gene to generate a 5 bp deletion and 1 pb insertion, respectively, and produce a non-functional mutant ROC5 protein. Oligoduplex based on both sgRNAs also has been designed.
Keyword: gene editing, leaf rolling, local cultivar, rice productivity, suboptimal land

Judul: Hamiltonian formulation for linear waves motion in two layers fluid
Abstrak: The surface wave could be considered as a wave that separates two fluids, namely water and air. Based on this assumption, it is introduced the interfacial wave, a wave between two layers of fluid with different density. The formulation of interfacial waves motion begins with deriving the base equation of irrotational ideal fluid. Furthermore, according to irrotational fluid assumption, the base equation can be stated in velocity potential. In this derivation, the fluids domain is assumed to be restricted by rigid lid boundary conditions, both at the upper and lower limit. Therefore, the interfacial waves motion can be explained in a hamiltonian formulation. In the hamiltonian formulation, total energy is defined as the sum of kinetic and potential energy. The hamiltonian system is obtained from reduction of kinetic energy by using the Dirichlet Neumann Operator. The resulted kinetic energy equation is nonlinear. Therefore, this form is linearized by first part of the Taylor expansion. This linearization gives a dispersion relation of linear wave. Based on this dispersion relation, the phase speed of the linear wave depends on the density ratio of the two layers fluid.
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