Teknik Penyuntingan Genom: CRISPR/Cas-9

Teknik Penyuntingan Genom: CRISPR/Cas-9

Kerangka Presentasi 1. Pendahuluan 2. CRISPR/Cas 3. Pemanfaatan Mekanisme CRISPR/Cas 4. Alur Proses CRISPR/Cas-9 5. Regulasi Produk CRISPR/Cas-9 6. Penutup

1. Pendahuluan Genome editing atau penyuntingan genom : Proses modifikasi gen/nukleotida fungsional di genom organisme Melibatkan enzim nuklease : Disruption (KO) Insertion Replacement

1. Pendahuluan Teknik penyuntingan genom: Zinc-Finger nucleases Nuklease FokI 3 kodon grup yang terikat Zn Transcription activator-like effector nucleases TALE akan menempel pada sekuens DNA secara spesifik

2. CRISPR/Cas CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats Cas : CRISPR-Associated Proteins (Nuclease) Mekanisme kekebalan adaptif bakteri dan archaea

2. CRISPR/Cas Short palindromic sequences CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats

2. CRISPR/Cas-9: Mekanisme Sistem alamiah bakteri (Tipe II): a. crrna (CRISPR RNA)/single guide RNA b. tracrrna (palindromic sequence) c. Cas9 protein (nuclease) crrna melekat pada gen target Nukleasae Cas9 akan membuat potongan pada daerah pelekatan crrna+dna genom (double strand break; DSB) DSB akan diperbaiki secara alami oleh perangkat sel target.

2. CRISPR/Cas-9: Mekanisme Mekanisme perbaikan DSB: a. NHEJ (Non-homologous End Joining) b. HR (Homologous Recombination)

3. Pemanfaatan Mekanisme CRISPR/Cas

4. Alur Proses CRISPR/Cas-9 a. Identifikasi sekuens gen target: Exon, Intron, Promoter b. Desain single guide RNA (sgrna) c. Kloning sgrna ke dalam plasmid replikasi d. Evaluasi hasil kloning e. Kloning sgrna ke dalam plasmid biner f. Evaluasi hasil kloning g. Transformasi/transfeksi konstruk gen ke organism target h. Seleksi dan Analisis fungsi

4. Alur Proses CRISPR/Cas-9: KO fungsi gen a. Identifikasi sekuen gen target: Exon, Intron, Promoter pada gene database. RAP-DB, SGN-DB, TAIR

4. Alur Proses CRISPR/Cas-9: KO fungsi gen b. Desain sgrna guide RNA (grna): terdiri dari 20 nt dari sekuen target dan 3 nt of PAM (protospacer-adjacent motif). Sekuen PAM: 5 -NGG dan 5 -NAG. 5 -NGG sangat sering digunakan. Untuk KO gen: menggunakan exon ke-1 Sekuen exon-1 diinput ke website CRISPR-P (http://cbi.hzau.edu.cn/cgibin/crispr).

4. Alur Proses CRISPR/Cas-9: KO fungsi gen 2 Kandidat sgrna 4 1 6 3 8 109 5 7 Seleksi kandidat grnas dari CRISPR-P higher match score value position of each grna mismatches are in non-exon region mismatch is preferable closed to PAM sequence (NGG) sequence (in a range of 12 bp after PAM) since it will unlikely contribute to offtarget mutation lower mismatch score value not contained restriction site used in the cloning

4. Alur Proses CRISPR/Cas-9: KO fungsi gen Seleksi kandidat grnas dari CRISPR-P higher match score value position of each grna mismatches are in non-exon region mismatch is preferable closed to PAM sequence (NGG) sequence (in a range of 12 bp after PAM) since it will unlikely contribute to off-target mutation lower mismatch score value not contained restriction site used in the cloning

4. Alur Proses CRISPR/Cas-9: KO fungsi gen Seleksi kandidat grnas dari CRISPR-P At least 3 grna candidates were submitted to Azimuth worksheet for predicting grna efficiency. Steps for submitting grna to Azimuth as follows: 30 nucleotides, consisting of 4 nt upstream sequence of grna, 23 nt grna+pam, and 3 nt downstream sequence of PAM were inputted in Azimuth worksheet. A value of -1 (ignored) was filled in parameters columns ( cutsite and percentpeptide ). The efficiency value was showed in predictedscore column. The grna with values of 0.6-1.0 is indicated to have higher efficiency and could be used for further step. Efficiency score of grna is 0.6-1.0 (high efficiency), 0.4-0.6 (grey zone), and <0.4 (low efficiency).

4. Alur Proses CRISPR/Cas-9: KO fungsi gen Ordering grna: grna should be ordered in a set of complementary oligo DNAs (forward and reverse sequences) Oligos consist of 20 nt target sequence (without PAM) and a restriction site linker (e.g. BbsI site: GTTG-<guide forward 20 bp>, AAAC-<guide reverse 20 bp>).

4. Alur Proses CRISPR/Cas-9 Kloning sgrna ke plasmid replikasi Antibiotic resistant Promoter specific GG grna (20 nt) Scaffold/gblock (80nt) Poly T PacI BbsI AscI

4. Alur Proses CRISPR/Cas-9 Kloning sgrna ke plasmid biner Antibiotic resistant Cas9 specific Promoter specific GG grna (20 nt) Scaffold/gblock (80nt) Poly T PacI BbsI AscI

4. Alur Proses CRISPR/Cas-9 Plasmid untuk konstruksi CRISPR/Cas-9

4. Alur Proses CRISPR/Cas-9 Evaluasi: Sekuensing Koloni PCR

5. Regulasi Produk CRISPR/Cas-9 Mushroom case US FDA European Regulation

5. Regulasi Produk CRISPR/Cas-9 Mushroom case US FDA European Regulation

5. Regulasi Produk CRISPR/Cas-9

Application on CRISPR/Cas-9 Product Elucidation on gene functions Genetic improvement: Plant, microbes Gene Therapy Synthetic Biology

6. Penutup Teknologi CRISPR/Cas-9 menyediakan teknologi yang lebih sederhana dan dapat digunakan untuk forward dan reverse genetics. Meskipun regulasi produk CRISPR/Cas-9 masih belum dipahami secara sama oleh berbagai pihak, teknologi ini dapat membantu pemahaman mekanisme regulasi molekuler/fungsi gen.

Acknowledgements JICA Satreps Alang-alang Dr. Satya Nugroho Dr. N.S. Hartati Lab of Metabolic Science of Forest Plants and Microorganism, RISH, Kyoto University Prof. T. Umezawa Dr. T. Miyamoto Dr. Rie Takada Technicians and Students