Lee, J. K. | bioRxiv Science

bioRxiv2017

Directed evolution of CRISPR-Cas9 to increase its specificity

The use of CRISPR-Cas9 as a therapeutic reagent is hampered by its off-target effects. Although rationally designed S. pyogenes Cas9 (SpCas9) variants that display higher specificities than the wild-type SpCas9 protein are available, these attenuated Cas9 variants are often poorly efficient in human cells. Here, we have used a directed evolution approach in E. coli to obtain Sniper-Cas9, which shows high specificities without sacrificing on-target activities in human cells.

biochemistry↗

bioRxiv2017

Abelson tyrosine-protein kinase 2 Regulates Myoblast Proliferation and Controls Muscle Fiber Length

Muscle fiber length is nearly uniform within a muscle but widely different among muscles. Here, we show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm and other muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of available myoblasts and leading to increased myoblast fusion. Abl2 acts in myoblasts, but expansion of the diaphragm muscle causes a reduction in size of the diaphragm central tendon. Replacement of tendon for muscle is likely responsible for the reduced stamina of abl2 mutant mice. Further, ectopic muscle islands, each composed of myofibers of uniform length and orientation, form within the central tendon in abl2+/- mice. Induction of scleraxis in tendon cells at the ends of these muscle islands suggests that myofibers stimulate tendon differentiation, which in turn regulates myofiber length.

developmental biology↗