New Additions to the CRISPR Toolbox: CRISPR-CLONInG and CRISPR-CLIP for Donor Construction in Genome Editing

Abstract

CRISPR-Cas has proven to be the most versatile genetic tinkering system of our time, predominantly as a precision genome editing tool. Here, we demonstrate two additions to the repertoire of CRISPRs application for constructing donor DNA templates. (i) CRISPR-CLONInG (CRISPR-Cutting & Ligation Of Nucleic acid In vitro via Gibson) was devised to enable efficient cut-and-paste of multiple complex DNA fragments by using CRISPR/Cas9 as a digestion alternative with precision and exclusivity features, followed by joining the digested products via Gibson Assembly, to rapidly construct dsDNA and AAV donor vectors without cloning scars. (ii) CRISPR-CLIP (CRISPR-Clipped Long ssDNA via Incising Plasmid) was devised as a DNA clipping tool to efficiently retrieve long single-stranded DNA (lssDNA) from plasmid, up to 3.5 kbases, which can be supplied as the donor template for creating genetically engineered mice via Easi-CRISPR. We utilized two different Cas types (Cpf1 and Cas9n) to induce two distinct incisions at the respective ends of the lssDNA cassette junctions on the plasmid, yielding three independent single-stranded DNA units of unique sizes eligible for strand separation, followed by target strand clip-out through gel extraction. The retrieval of the lssDNA donor circumvents involvements of restriction enzymes and DNA polymerase-based steps, hence not only retains sequence fidelity but carries virtually no restriction on sequence composition, further mitigating limitations on current Easi-CRISPR method. With the add-on feature of universal DNA-tag sequences of Cpf1-Cas9 duo PAM, CRISPR-CLIP can be facile and applicable to generate lssDNA templates for any genomic target of choice. Additionally, we demonstrate robust gene editing efficiencies in neuroblastoma cell line as well as in mice attained with the AAV and lssDNA donors constructed herein.