STARSET_MirrorNew CRISPR-Cas approach permits more precise DNA cleavage
CRISPR-Cas editing has transformed the ability of researchers to alter DNA—for example, to cleave specific DNA sequences in ways not possible with restriction enzymes, or proteins isolated from bacteria that have been used for decades to cleave DNA sequences at specific sites. Although CRISPR-Cas tools can be programmed to target and cut virtually any DNA sequence, a major constraint in their targeting is the requirement to first recognize a short sequence flanking the target called a protospacer adjacent motif (PAM). Therefore, DNA could previously only be cut at sites flanking this specific motif.
CRISPR-Cas编辑已经改变了研究人员改变DNA的能力——例如，用限制性内切酶或从细菌中分离的蛋白质无法做到的方式切割特定的DNA序列，而这种蛋白质已经被用于切割特定位点的DNA序列几十年了。尽管CRISPR-Cas工具可以被编程定位和切割几乎任何DNA序列，但其定位的一个主要限制是要求首先识别位于目标侧翼的短序列，称为原间隔相邻基序(PAM)。因此，DNA以前只能在这个特定基序的侧面被切割。
In this latest research, the team that previously engineered a nearly PAMless CRISPR–Cas9 variant, named SpRY, tested its utility to serve as a universal DNA cleavage tool.
在这项最新的研究中，该团队之前设计了一个几乎没有pam的CRISPR-Cas9变体，名为SpRY，测试了其作为通用DNA切割工具的效用。
By designing SpRY and guide RNA (gRNA) complexes that targeted more than 130 DNA sequences in laboratory experiments, the scientists surprisingly discovered that SpRY is PAMless in vitro and can effectively cleave DNA at any sequence programmed by the gRNA. The investigators also showed that their technology can overcome limitations of restriction enzymes.
通过设计SpRY和导向RNA (gRNA)复合体，在实验室实验中靶向超过130个DNA序列，科学家们惊奇地发现，SpRY在体外是无PAMless的，可以有效地切割由gRNA编程的任何序列的DNA。研究人员还表明，他们的技术可以克服限制性内切酶的局限性。
“We demonstrate that SpRY DNA digests—or SpRYgests—enable DNA cutting at practically any sequence, including a wide range that were previously untargetable with restriction enzymes or other CRISPR-Cas proteins,” says senior author Benjamin Kleinstiver, Ph.D., an Assistant Investigator at the Center for Genomic Medicine at Mass General Hospital and an Assistant Professor at Harvard Medical School. “This new method permits researchers to cut DNA in a test tube at any DNA location of choice. The new capabilities offered by SpRYgests will accelerate and reduce the cost of various basic research applications, including for studies that could have eventual clinical implications.”
“我们证明，SpRY DNA消化器——或称sprygests——能够在几乎任何序列上切割DNA，包括以前用限制性内切酶或其他CRISPR-Cas蛋白质无法靶向的广泛序列，”高级作者Benjamin kleinstver博士说，他是麻省总医院基因组医学中心的助理研究员和哈佛医学院的助理教授。“这种新方法允许研究人员在试管中选择任何DNA位置切割DNA。SpRYgests提供的新功能将加快并降低各种基础研究应用的成本，包括可能最终产生临床影响的研究。”
The researchers envision that SpRYgests could be widely applicable to simplify typical molecular cloning approaches, for more complex cloning methods, for assembling next-generation sequencing libraries, and many others.
研究人员设想，SpRYgests可以广泛应用于简化典型的分子克隆方法、更复杂的克隆方法、组装下一代测序库和许多其他方法。
Read more at Phys.org
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