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.

中文翻译：

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CRISPR工具箱的新功能：用于基因组编辑中供体构建的CRISPR-CLONInG和CRISPR-CLIP。

事实证明，CRISPR-Cas是当今时代最通用的基因修补系统，主要是作为精密的基因组编辑工具。在这里，我们展示了CRISPR在构建供体DNA模板中的应用程序库中的两个新增功能：CRISPR- CLONInG和CRISPR- CLIP。CRISPR-克隆（CRISPR- ç的Utting和大号igation ö ˚F Ñ ucleic酸在体外通过ģibson）旨在通过使用CRISPR-Cas9作为具有精确和排他性功能的消化替代品，然后通过Gibson Assembly加入消化产物来构建双链DNA和腺苷，从而有效剪切和粘贴多个复杂的DNA片段。 -相关病毒（AAV）供体载体快速克隆而无疤痕。CRISPR- CLIP（CRISPR- ç唇形大号经由翁的ssDNA我ncising P lasmid）被设计作为DNA裁剪工具有效地从质粒中检索长的单链DNA（lssDNA），至多3.5 kbase，其可以作为供体来提供通过Easi创建基因工程小鼠的模板-CRISPR。我们利用两种不同的Cas类型（Cpf1和Cas9n）在质粒的lssDNA盒连接的各个末端诱导了两个不同的切口，产生了具有大小适合进行链分离的三个独立的单链DNA单元，随后是靶链片段通过凝胶提取。lssDNA供体的检索规避了限制酶和基于DNA聚合酶的步骤的参与。因此，它不仅保留序列的保真度，而且进行序列组成几乎没有限制，对当前进一步缓解局限性爱莎-CRISPR方法。具有Cpf1-Cas9二重原型间隔子相邻基序的通用DNA标签序列的附加功能，CRISPR- CLIP可以很容易地用于生成任何选择的基因组靶标的lssDNA模板。另外，我们证明了在神经母细胞瘤细胞系以及用本文构建的AAV和lssDNA供体获得的小鼠中强大的基因编辑效率。