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

The CRISPR Journal Volume 3, Number 2, 2020 Mary Ann Liebert, Inc. DOI: 10.1089/crispr.2019.0062 RESEARCH ARTICLE New Additions to the CRISPR Toolbox: CRISPR-CLONInG and CRISPR-CLIP for Donor Construction in Genome Editing Dorjee T.N. Shola,1,* Chingwen Yang,1 Vhy-Shelta Kewaldar,1 Pradip Kar,1 and Victor Bustos2 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 CRISPR’s application for constructing donor DNA templates: CRISPR-CLONInG and CRISPR-CLIP. CRISPR-CLONInG (CRISPR-Cutting and 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 construct double-stranded DNA and adeno-associated virus (AAV) donor vectors rapidly without cloning scars. CRISPR-CLIP (CRISPR-Clipped Long ssDNA via Incising Plasmid) was devised as a DNA clipping tool to retrieve long single-stranded DNA (lssDNA) efficiently from plasmid, up to 3.5 kbase, 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, it not only retains sequence fidelity but also carries virtually no restriction on sequence composition, further mitigating limitations on the current Easi-CRISPR method. With the add-on feature of universal DNA-tag sequences of Cpf1-Cas9 duo protospacer adjacent motif, 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 the neuroblastoma cell line, as well as in mice attained with the AAV and lssDNA donors constructed herein. Introduction The versatility of class 2 CRISPR system is attributable to the simplicity of Cas nuclease being guided by a single programmable RNA1 coupled with a unique spacer sequence for precise target navigation. The commonly used CRISPR protein, SpCas9, recognizes a protospacer adjacent motif (PAM) NGG, which exists once in every 42 bases in the human genome.2 In addition, a mutant version from xCas9 group recognizes an even shorter PAM ‘‘NG,’’3 along with Cpf1 for AT-rich sequences,4 these Cas variants further relax the PAM stringency to allow its binding to all four nucleotides of the DNA sequence. Together with Cas9’s stability and ATP independent catalytic reaction for facilitating DNA cleavage,5 the 1 CRISPR system has attracted a wide variety of applications, prevalently as a robust tool for precision genome engineering in mammalian cells.6,7 CRISPR-mediated genome editing is carried out by using RNA-guided Cas9 to induce a DNA break at the genomic locus of interest, followed by harnessing an innate DNA repair mechanism to create indels for variants of gene disruptions or genetic modifications with defined outcomes. The latter is attained by additionally supplying an exogenous DNA template that carries the desired sequence flanked by homology arms (HA) to CRISPR cut site(s), and through the homology-directed repair (HDR) pathway, desired modifications can be precisely integrated into the genome of target organisms in a highly CRISPR and Genome Editing Resource Center and 2Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York, USA. *Address correspondence to: Dorjee T.N. Shola, PhD, CRISPR and Genome Editing Resource Center, The Rockefeller University, 1230 York Ave, New York, NY 10065, Email: ª Dorjee T.N. Shola et al. 2020; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 109 110 efficient manner. To generate genetically engineered mouse models, donor templates can be supplied as either single-stranded oligodeoxynucleotides (ssODN) or doublestranded DNA (dsDNA) vectors.8 The former functions as an efficient donor in zygotes, yet imposes length limitations of *200 bases due to technical difficulties in chemical synthesis, making it suitable only for minor genetic alterations (<50 bp), whereas dsDNA-mediated editing can accommodate larger-scale genetic modifications (up to 10 kb or even longer), which has been routinely conducted via mouse embryonic stem cells (mESCs) due to poor efficiency in zygotes. Recent development of Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) has successfully expanded the use of single-stranded DNA donor up to *2 kbase long, referred to as long single-stranded DNA (lssDNA), to introduce modifications over much larger genomic regions in mouse and rat zygotes, as well as in human T cells.9–11 The length capacity of lssDNA suffices for most genome editing purposes that used to be mediated via the mESC route, whereby the timeline for generating the founder mice (F0) can be accelerated to as little as 2 months. The construction of dsDNA donors commonly relies on BAC recombineering or multi-step cloning methods to ligate a vector backbone and multiple DNA fragments, which often need to be acquired from various existing plasmids through the use of polymerase chain reaction (PCR) amplification and restriction enzyme (RE). The development of seamless cloning methods, such as Gibson Assembly,12 allows multiple DNA components to be assembled into a custom donor vector in a single step in vitro, averting any footprint and incorrect insert orientation, offering a rapid alternative to the lengthy and laborious conventional approach for donor template assembly. Nonetheless, seamless cloning requires each of the assembly components to carry complementary overhang sites (e.g., type IIS RE sites and Gibson overhangs for Golden Gate and Gibson Assembly, respectively), which are routinely created by PCR amplification. Such a process tends to stumble over DNA sequences with extended length or complexity, a common scenario when amplifying vector backbones with highly repetitive or palindromic sequences (e.g., multiple Lox sites in FLEx vector or ITR sequences in adeno-associated virus [AAV] vector), hence hindering the vector cloning. To address these PCR issues, especially in acquiring vector backbones, we utilize CRISPR-Cas9 to replace RE as the digestion tool to excise undesired DNA segm (...truncated)