Simultaneous Delivery of CRISPR/Cas and Donor DNA using Cell‐Penetrating Peptide‐Adaptors

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are utilized as a form of adaptive immunity in prokaryotes. CRISPR detects and eliminates foreign nucleic acids such as those found in bacteriophage. To achieve this form of ‘memory of infection,’ CRISPR associated proteins (Cas) form complexes with RNA to cause double-stranded breaks (DSBs) in the invader's sequence. The invader's DNA between these breaks is incorporated within the CRISPR locus in the bacterial genome. CRISPR/Cas9 has been a breakthrough for genome editing due to its specificity and potential to allow genome editing in patients, where it could be used to treat many diseases such as cancer or hemophilia. The present work aims to enhance CRISPR efficiency by delivery of Cas9 complexed with guide RNA (crRNA and tracRNA) to the nuclei of living cells using a novel cell-penetrating peptide (CPP)-adaptor system. CPPs have long been used to deliver biomolecules to living cells, but CPP-cargos remained trapped in endosomes. To solve this problem, we used a novel CPP adaptor, TAT-CaM, a fusion protein in which the CPP from HIV TAT is fused to human calmodulin (CaM). When Ca2+ is present, TAT-CaM binds to an engineered calmodulin binding site (CBS) on cargo proteins (e.g. CBS-Cas9). After endocytosis, TAT-CaM dissociates from cargos as Ca2+ concentrations drop during trafficking. Like other CPPs, TAT-CaM remains trapped in endosomes but cargos efficiently escape. In the present study, biolayer interferometry (BLI) and confocal microscopy were used to demonstrate Ca2+-dependent binding and delivery of CRISPR complexes to the nuclei of cells. We have employed a similar scheme to deliver in parallel donor DNA that can be used by the cell's homologous recombination machinery to effect editing. Donor DNA is internalized simultaneously with CRISPR/Cas using a CBS-tamavidin fusion protein that binds both TAT-CaM and biotinylated donor DNA. Preliminary results show that CBS-tamavidin is wellexpressed in E. coli and readily purified using immobilized metal affinity chromatography. BLI analysis showed that CBS-tamavidin binds TAT-CaM affinity in the presence of Ca2+ while simultaneously binding biotinylated DNA, both with high affinity (fast on/slow off). Confocal microscopy showed that CBS-Cas9 and CBS-tamavidin/biotin-DNA enter cells in the presence TAT-CaM but not in the absence. Support or Funding Information National Institutes of Health grant GM R15120691 Preliminary results for delivery of CBS-Cas9 in Baby Hamster Kidney(BHK) cells using confocal microscopy. CRISPR RNAs are labeled with Alexa 550 red, the nucleus is labeled with NucBlue, the Cas9 and TAT CaM are unlabeled. The first column only shows the CRISPR RNA, the second column only shows the nucleus and the third column shows the composite of the first and the second column. The first row and the second row show the cells with CRISPR complex in 10 and 20 minutes respectively. BioLayer Interferometry (BLI) analysis of CBS-Cas9. (A) Association and dissociation of CBS-Cas9 (analyte at concentrations starting from 200nM to 50nM) in TAT CaM(ligand at 1uM) and a calcium-containing buffer respectively. (B) CBS-Cas9 dissociation in EDTA(chelates calcium) from 200nM to 50nM. (C) Kinetic parameters for CBS-Cas9 from A and B. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.