Efficient genome editing by CRISPR-Mb3Cas12a in mice

Abstract

As an alternative and complementary approach to Cas9-based genome editing, Cas12a has not been widely used in mammalian cells largely due to its strict requirement for the TTTV protospacer adjacent motif (PAM) sequence. Here, we report that Mb3Cas12a (Moraxella bovoculi AAX11_00205) can efficiently edit the mouse genome based on the TTV PAM sequence with minimal numbers of large on-target deletions or insertions. When TTTV PAM sequencetargeting CRISPR (cr)RNAs of 23 nt spacers are used, >70% of the founders obtained are edited. Moreover, the use of Mb3Cas12a tagged to monomeric streptavidin (mSA) in conjunction with biotinylated DNA donor template leads to high knock-in efficiency in two-cell mouse embryos, with 40% of founders obtained containing the desired knock-in sequences.

Ablation of the miR-465 Cluster Causes a Skewed Sex Ratio in Mice

“…Like the miR-465 cluster, miR-892b is also flanked by Slitrk2 and Fmr1 on the X chromosome ( 14 ). To define their physiological roles, we deleted the entire miR-465 cluster in the mouse genome using CRISPR-Cas9 ( Figures 1A ; S1A ), as previously described ( 14 , 18 , 19 ). PCR genotyping and Sanger sequencing confirmed that the genomic loci of these miRNAs were successfully deleted ( Figures S1B, C ).…”

“…The animal use protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Nevada, Reno (Protocol# 00494). Generation of global miR-465 KO mice and mouse genotyping were performed as described ( 14 , 18 , 19 ). gRNA and genotyping primers are listed in Table S1 .…”

A sexual dimorphic role of themiR-465cluster in placental development

“…To define their physiological roles, we deleted the entire miR-465 cluster using CRISPR-Cas9 ( Fig. S1), as previously described [2,6,7]. The miR-465 KO mice were fertile, have normal testis size (Fig.…”

“…Generation of global KO mice and mouse genotyping were performed as described [2,6,7]. All gRNA and genotyping primers were listed in Table S3.…”

miRNA-dependent poly(A) length control in uncoupling transcription and translation of haploid male germ cells

“…The animal protocols was approved by the Animal Care and Use Committee of Nantong University or University of Nevada, Reno. Male germ cell-specific Drosha conditional KO mice (Drosha loxp/loxp mice were bred with Stra8-iCre mice) and global KO of X linked miR-506 family were generated and genotyped at the University of Nevada, Reno as previously described [23,46,48,75].…”