Can CRISPRcas 9 editing of APP Cterminus promote

Can CRISPR/cas 9 editing of APP C-terminus promote the amyloidogenic pathway? By Laura Wyckaert Sun J 1, Carlson-Stevermer J 2, 3, Das U 4, Shen M 5, Delenclos M 6, Snead AM 7, Koo SY 7, Wang L 1, Qiao D 1, Loi J 8, Petersen AJ 5, Stockton M 5, Bhattacharyya A 5, Jones MV 8, Zhao X 5, 8, Mc. Lean PJ 6, Sproul AA 7, 9, Saha K 2, 3, Roy S 10, 11, CRISPR/Cas 9 editing of APP C-terminus attenuates β-cleavage and promotes α-cleavage, Nature Communications, 2019 Jan 3; 10(1): 53. doi: 10. 1038/s 41467 -018 -07971 -8.

Alzheimer’s Disease • Degenerative disease: causes dementia • Progressive memory loss and cognitive dysfunction • β-amyloid plaques and hyperphosphorylated forms of the protein Tau

The amyloid cascade hypothesis • Plaques: amyloid-β peptides • Derive from APP • plaques ▫ Pathological trigger for a cascade ▫ Crucial in AD development

The APP metabolism • APP can get cleaved in two different ways • Non-amyloidogenic: α-secretase -> neuroprotective • Amyloidogenic: β-secretase -> Aβ peptide

YENPTY-motif • C-terminus of APP • Mediates internalization of APP into the endosomes • Promotes amyloidogenic pathway • N-terminus: axonal growth • Target of our research • APLP 1 and APLP 2: compensate

Aims • To identify PAM (protospacer adjacent motif) sites at the APP Cterminus conserved in both human and mouse. • To design a single-guide RNA that guides the cas 9 to the C-terminus of APP in the host genome. • To examine the effects of CRISPR/Cas 9 editing on neurophysiology • To evaluate the effect of APP C-terminus editing on the amyloidogenic pathway in vivo.

Methods- In vitro • Step 1: Øfind target sequences in C-terminus ØIndentify PAM sites Ødesign sg. RNAs ØGenome sequencing tools • Step 2: ØMice-derived neuro 2 a cells ØLentiviral vectors carrying sg. RNA ØCompare efficiency

Methods- In vitro • Step 3: Test if the APP-editing is successful Ø Y 188 antibody recognizes C-terminus Ø in western blot and immunofluorescence tests Ø Genome deep sequencing : frequency of base-pair matches Ø Will also do test in IPS cell line carrying the London mutation • Step 4: examine the effects of CRISPR/Cas 9 editing on neurophysiology Ø cultured hippocampal neurons Ø Neurite outgrowth and synaptic function • Step 5: In vivo Ø injection of AAV vectors in mouse hippocampi

Expected results • Increased levels of the s. APP α: upregulation of non amyloidogenic pathway • Less secretion of the amyloid β protein: inhibition of the amyloidogenic pathway

Significance • Until now focus of CRISPR/Cas 9 gene editing until now: gene deletion or correction of mutation • Approaches of limited use in neurodegenerative disorders • the start of the wide-spread use of CRISPR/Cas 9 -guided gene editing in neurodegenerative diseases • 150 million people affected • Still no cure available

Thank you so much for listening ! Sun J 1, Carlson-Stevermer J 2, 3, Das U 4, Shen M 5, Delenclos M 6, Snead AM 7, Koo SY 7, Wang L 1, Qiao D 1, Loi J 8, Petersen AJ 5, Stockton M 5, Bhattacharyya A 5, Jones MV 8, Zhao X 5, 8, Mc. Lean PJ 6, Sproul AA 7, 9, Saha K 2, 3, Roy S 10, 11, CRISPR/Cas 9 editing of APP C -terminus attenuates β-cleavage and promotes α-cleavage, Nature Communications, 2019 Jan 3; 10(1): 53. doi: 10. 1038/s 41467 -018 -07971 -8.