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New 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.


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.


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.