Efficient CRISPR/Cas9 mediated large insertions using long single-stranded oligonucleotide donors in C. elegans.

Highly efficient generation of deletions, substitutions, and small insertions (up to ~ 150 bp) into the Caenorhabditis elegans genome by CRISPR/Cas9 has been facilitated by the use of single-stranded oligonucleotide donors as repair templates. However, insertion of larger sequences such as fluorescent markers and other functional domains remains challenging due to uncertainty of optimal performance between single-stranded or double-stranded repair templates and labor-intensive as well as inefficient protocols for their preparations. Here, we simplify the generation of long ssDNA as donors in CRISPR/Cas9. High yields of ssDNA can be rapidly generated using a standard PCR followed by a single enzymatic digest with lambda exonuclease. Comparison of long ssDNA donors obtained using this method to dsDNA demonstrates orders of magnitude increased insertion frequency for ssDNA donors. This can be leveraged to simultaneously generate multiple large insertions as well as successful edits without the use of selection or co-conversion (co-CRISPR) markers when necessary. Our approach complements the CRISPR/Cas9 toolkit for C. elegans to enable highly efficient insertion of longer sequences with a simple, standardized, and labor-minimal protocol.

MiR-35 buffers apoptosis thresholds in the C. elegans germline by antagonizing both MAPK and core apoptosis pathways.

Apoptosis is a genetically programmed cell death process with profound roles in development and disease. MicroRNAs modulate the expression of many proteins and are often deregulated in human diseases, such as cancer. C. elegans germ cells undergo apoptosis in response to genotoxic stress by the combined activities of the core apoptosis and MAPK pathways, but how their signalling thresholds are buffered is an open question. Here we show mir-35-42 miRNA family play a dual role in antagonizing both NDK-1, a positive regulator of MAPK signalling, and the BH3-only pro-apoptotic protein EGL-1 to regulate the magnitude of DNA damage-induced apoptosis in the C. elegans germline. We show that while miR-35 represses EGL-1 by promoting transcript degradation, repression of NDK-1 may be through sequestration of the transcript to inhibit translation. Importantly, dramatic increase in NDK-1 expression was observed in cells about to die. In the absence of miR-35, increased NDK-1 activity enhanced MAPK signalling that lead to significant increases in germ cell death. Our findings demonstrate that NDK-1 acts upstream of (or in parallel to) EGL-1, and that miR-35 targets both egl-1 and ndk-1 to fine-tune cell killing in response to genotoxic stress.