长单链DNA (ssDNA) 合成服务

1. CRISPR技术进行基因的插入、置换和修正

CRISPR/Cas9技术可在靶位点进行精准的切割，在靶位点产生双链断裂（double stranded breaks, DSBs），引导RNA（single guided RNA, sgRNA）与Cas9蛋白形成复合物后，可以识别靶位点相邻的PAM序列，然后Cas9蛋白可以发挥其核酸内切酶的功能，从而切割并在靶位点产生双链断裂。此时，有两种机制可以触发DNA修复，一种是非同源末端连接（non-homologous end joining, NHEJ），这种方法可能在双链断裂的位点引入突变。另一种机制是同源重组修复（HDR），这种方法通将修复模板中的待敲入DNA片段插入断裂的位点，从而来实现基因中DNA片段的敲入。

传统方法使用双链DNA（double stranded DNA, dsDNA）作为HDR中DNA片段的修复模板，但是最近的研究显示，利用CRISPR技术进行基因插入、置换和修正时，单链DNA（ssDNA或ssOND）是最合适的HDR DNA片段的供体模板。和dsDNA作为供体模板比较，使用ssDNA可以显著提高编辑效率和特异性，并且降低脱靶效应，细胞毒性和随机整合的风险也较之dsDNA大大降低，特别适合原代细胞、干细胞的基因编辑，基因改造动物模型的建立。

2. 体外转录

体外转录法（in vitro transcription , IVT）是在通过采用体外的DNA模板，利用RNA噬菌体聚合酶(T7, T3 或SP6)，获得转录产物RNA分子。三种不同类型的DNA模板可以用于IVT，环状质粒形式的DNA、PCR扩增后的DNA片段和单链DNA。转录产物RNA可以用于化学研究、基因学研究、结构研究等多个领域。同时，例如适体、核酶等人工功能性核酸也可以通过IVT的方法得到。

讲座视频：

• CRISPR技术编辑T细胞：利用非病毒的HDR模板对人类T细胞进行大量片段敲入
• 高效获得CRISPR KO/KI细胞系的策略解析

表明ssDNA作为更优CRISPR 基因编辑模板的文献：

ssDNA与dsDNA比较，可以显著提高编辑效率和特异性，较少脱靶效应，特别适合原代细胞、干细胞的基因编辑，基因改造动物模型的建立。

• "We recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement." Miura H, et al., Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors. Nat Protoc. (2018) 13(1):195-215.
• "We report that targeted chromosomal translocations are generated more efficiently when the all-in-one plasmid, RNP complex, and ssODN-based approaches are used, with the most efficient strategy being the combination of RNP complexes with translocation-ssODNs." Torres-Ruiz, et al., Efficient Recreation of t(11;22) EWSR1-FLI1+ in Human Stem Cells Using CRISPR/Cas9. Stem Cell Reports. (2017) 8: 1408–1420.
• "ssDNA templates have a unique advantage in terms of repair specificity while dsDNA donors can lead to a high rate of off-target integration." Li, et al., Design and specificity of long ssDNA donors for CRISPR-based knock-in. bioRxiv 178905; doi: https://doi.org/10.1101/178905.
• "The PITCh approach required 265 zygotes whereas Easi-CRISPR used only 105 zygotes, and the PITCh approach produced 33% correctly targeted pups, whereas Easi-CRISPR (using ssDNA donors) produced 100% correctly targeted pups." Quadros, et al., Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biology (2017) 18:92.
• "Similar to what has been described with viral HDR templates, we found evidence to suggest that double-stranded templates could integrate independent of target homology, albeit at low rates. These rare events could be reduced almost completely by using single-stranded DNA (ssDNA) templates." Roth, et al., Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 559 (2018) 405–409.