IMPROVEMENT OF CRISPR GENE EDITING EFFICIENCY AND BEYONDS

Transcription

1 IMPROVEMENT OF CRISPR GENE EDITING EFFICIENCY AND BEYONDS YONGLUN LUO (ALUN) VIB, NOV Associate Professor, Department of Biomedicine, Aarhus University, Denmark Executive Director, Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, China AU-iCRISPR

2 THE ERA OF GENE EDITING PRECISION GENE EDITING DELETION (KNOCKOUT) INSERTION (KNOCKIN) SUBSTITUTION (MUT/COR) REGULATION (CIS/TRANS) MODIFICATIONS (EPIGENETIC)

3 Programmable, precision gene editing tools: Genome Position System (GPS) precision positioning, cleavage, and modification ZFN TALEN CRISPR-Cas9

4 Cas9 grna The CRISPR-Cas9 gene editing system is composed of two functional elements: a Cas9 DNA endonuclease and a programmable chimeric guide RNA (grna). grna is composed of a user designed guide sequences, normally 20 nt, and a scaffold RNA sequences. The Cas9 protein is positioned by grna to any doublestranded target DNA site which is complimentary to the grna guide sequences and have a unique DNA motif (protospacer adjacent motif, PAM). The Cas9 nuclease activity is activated and introduced a double-strand DNA break to the target site. DNA PAM indels Cytoplasm Nucleus

5 DOUBLE STRAND DNA BREAKS (DSB) FACILITATE GENE EDITING Precise and programmable generation of double-strand DNA breaks stimulate DNA repair double strand DNA breaks DSBs in mammalian cells are prone to the NHEJ-MMEJ-SSA repair pathway (error prone) DSB repair by HDR in mammalian cells is relatively low compared to NHEJ, less than 1% (precision) NHEJ-MMEJ-SSA deletions/insertions EFFICIENCY SPECIFICITY homology-directed repair targeted insertion/ mutation/correction

6 CRISPR TOOL BOX AND APPLICATIONS IN THE DREAM TEAM CRISPR-CAS9 SYSTEMS All-in-one plasmid system, PX330 puromycin, both SpCas9 and SpCas9nickase All-in-one lentiviral expression system (puro), or single expression system, SpCas9 (bla), grna (puro) All-in-one piggybac system (puro), SpCas9 All-in-one AAV system, SaCas9 Recombinant SpCas9 with enhanced efficiency CRISPR screening CRISPR array (5000 genes, ongoing) Plasmid multiplex grna expression system, plasmid, up to 30 grnas (Johan) C-check system, quantification of grna activity and enrichment of gene edited cells (Yan) Repeat-edit system (LIN, on-going, Lundbeckfond) STOP system (AU-BGI group) AAV multiplex grna expression system (AU-BGI group) Rainbow system (AU-BGI group) CRISPRi CRISPRa CRISPRme APPLICATIONS Pig modeling: - HIP pig - Diabetes - Rainbow pig - Development - PERV KO - XT - pgecko - XT Gene editing in human cells: Over 100 KO lines generated Over 5 KI lines generated

7 Niu et al., Science 357, (2017) Lin Lin

8 1. Enhance CRISPR gene editing by fusing Cas9 protein to E.coli RecA dcas9-grna Cas9-gRNA single-tethered DNA curtains assay Steinberg et al. Nature 507, (06 March 2014) Our hypothesis is that : Fusing wild-type Cas9 to DSB repair protein or domains can tune the choice of DSB repair

9 CAS9-RECA FUSION ENHANCES CRISPR-MEDIATED INTEL EFFICIENCY Lin L. et al. J Biotechnol Apr 10;247:42-49.

10 CAS9-RECA ENHANCES CRISPR-MEDIATED GENE DELETION Lin L. et al. J Biotechnol Apr 10;247:42-49.

11 C-CHECK: A GENE EDITING REPORTER SYSTEM BASED ON SINGLE STRAND MEDIATED DSB REPAIR A system based on double-strand DNA repair mechanism in cells System highlights: Rapid: 3 days Sensitive: 1% Multiplexed: up to 30 grna Compatible with FACS Compatible with high-content screening Zhou et al. Cell Mol Life Sci Jul;73(13):

12 CAS9-RECA ENHANCES CRISPR GENE EDITING EFFICIENCY VIA FAVORING SSA-MEDIATED DSB REPAIR WHILE REPRESSING HDR Lin L. et al. J Biotechnol Apr 10;247:42-49.

13 Gene A Gene C Gene B Gene E Gene D Gene F Simultaneous, multiplexed gene editing?

14 3. MULTIPLEXED GENE EDITING T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 Single grna expression vector M10 grna array [Johan Vad-Nielsen et al CMLS]

15 APPLICATIONS Lin L. et al. Oncotarget [Johan Vad-Nielsen et al CMLS]

16 Submitted for publication. Nielsen JP. et al. 2017

17 GENE EDITING SOLUTIONS IN OUR GROUP DAPI/STAT3-GFP/lysosome CRISPR-mediated gene tagging (submitted), Liu et al CRISPR-mediated large deletion (on-going), Signe Multiplexed editing. (Lin and Johan). CMLS 2017, Bio-protocols 2017, Oncotarget 2017, Gene activation (CRISPRa) Xiong K. et al. Cell Rep (3): p Johan. Submitted. CRISPR epigenome editing Lin et al. Submitted. CRISPR Gene-trace Lin et al. on-going

18 CRISPR gene editing is now successfully applied in drug development, animal modeling, genetic screening, novel biomaterials, food enhancement, bio-fuel production, gene therapy etc.

19 Write Edit Cells Animals Read A few facts by the end of 2018: One HQ pig facility Five key platforms Ten senior PIs 20 students (master, PhD) 100 staffs 100TB NGS data

20 BGI-Qingdao Overview Ø BGI-Qingdao Park We have constructed 3 buildings in BGI-Qingdao park at phase I with totally of 20,000 lab and office area. Stage II #1 building Super-computing facility Stage I #2 building Research Platform; Office #3 building Biobank; Animal experiment lab; Sequencing Platform