Tissue-Specific Gene Inactivation inXenopus laevis: Knockout oflhx1in the Kidney with CRISPR/Cas9

Abstract

Studying genes involved in organogenesis is often difficult because many of these genes are also essential for early development. The allotetraploid frog, , is commonly used to study developmental processes, but because of the presence of two homeologs for many genes, it has been difficult to use as a genetic model. Few studies have successfully used CRISPR in amphibians, and currently there is no tissue-targeted knockout strategy described in The goal of this study is to determine whether CRISPR/Cas9-mediated gene knockout can be targeted to the kidney without perturbing essential early gene function. We demonstrate that targeting CRISPR gene editing to the kidney and the eye of F0 embryos is feasible. Our study shows that knockout of both homeologs of results in the disruption of kidney development and function but does not lead to early developmental defects. Therefore, targeting of CRISPR to the kidney may not be necessary to bypass the early developmental defects reported upon disruption of Lhx1 protein expression or function by morpholinos, antisense RNA, or dominant negative constructs. We also establish a control for CRISPR in by editing a gene () that when knocked out results in albinism without altering kidney development. This study establishes the feasibility of tissue-specific gene knockout in , providing a cost-effective and efficient method for assessing the roles of genes implicated in developmental abnormalities that is amenable to high-throughput gene or drug screening techniques.

The Role of the Wnt/PCP Formin Daam1 in Renal Ciliogenesis

“…Wild type oocyte-positive X. laevis adults were purchased from Nasco (LM00531MX) and embryos were obtained from these adults and reared as previously described (DeLay et al , 2018). This protocol was approved by the University of Texas McGovern Medical School Institutional Animal Care and Use Committee (IACUC) (protocol #: AWC-16-0111).…”

Dynamin binding protein is required forXenopus laeviskidney development

“…A sgRNA against slc45a2 was generated for use as a control . DNA templates for sgRNAs were produced by PCR, and T7 polymerase was used to transcribe sgRNA from the DNA templates as previously described (Bhattacharya et al, 2015;DeLay et al, 2018). For long-term storage, sgRNA was diluted to 1000 ng/µL and kept at -80 °C.…”

“…Cas9 protein (CP01; PNA Bio) and sgRNA were incubated together at room temperature for at least 5 min prior to microinjection . MOs, RNA constructs and Cas9/sgRNAs were co-injected with either membrane-RFP RNA, Alexa Fluor 488 fluorescent dextran or rhodamine dextran as a tracer (Davidson et al, 2006;DeLay et al, 2018;DeLay et al, 2016).…”

“…Additionally, the Xenopus tadpole epidermis is transparent and the kidney is located just under the epidermis, allowing visualization of the pronephros without dissection . It is also possible to easily modulate gene expression in Xenopus embryos through overexpression, knockdown and knockout experiments via microinjection of RNA constructs, antisense morpholino oligonucleotides (MOs) and CRISPR constructs DeLay et al, 2018;Miller et al, 2011). The established cell fate maps of the early Xenopus embryo facilitate tissue-targeted modulation of gene expression by microinjection into the appropriate blastomere DeLay et al, 2016;Moody, 1987a;Moody, 1987b).…”

SLC45A2 protein stability and regulation of melanosome pH determine melanocyte pigmentation

“…membrane associated transporter protein, MATP or antigen isolated from immunoselected melanoma-1, AIM1) (Newton, Cohen-Barak et al, 2001). Mutations in the homologous gene underlie pigment dilution in a number of vertebrate species, including gorilla, several breeds of dog, tigers, horses, mice, shrew, chickens, pigeons, quail, frogs, fish and perhaps cattle (Caduff, Bauer et al, 2017, DeLay, Corkins et al, 2018, Domyan, Guernsey et al, 2014, Dooley, Schwarz et al, 2013, Fukamachi, Shimada et al, 2001, Gunnarsson, Hellström et al, 2007, Mariat, Taourit et al, 2003, Minvielle, Cecchi et al, 2009, Newton et al, 2001, Prado-Martinez, Hernando-Herraez et al, 2013, Rothammer, Kunz et al, 2017, Tsetskhladze, Canfield et al, 2012, Tsuboi, Hayashi et al, 2009, Wijesena & Schmutz, 2015, Winkler, Gornik et al, 2014, Xu, Dong et al, 2013, and polymorphisms at the SLC45A2 locus are associated with skin tone differences and skin aging in several human population studies (Adhikari et al, 2019, Branicki et al, 2008, Cerqueira, Hunemeier et al, 2014, Fracasso, de Andrade et al, 2017, Han et al, 2008, Jonnalagadda, Norton et al, 2016, Law, Medland et al, 2017, Liu et al, 2015, Lopez, Garcia et al, 2014, Soejima & Koda, 2007, Stokowski et al, 2007, Yuasa, Umetsu et al, 2006. OCA4 patients have very low levels of pigmentation and phenotypically resemble OCA2 patients who lack the melanosomal chloride channel, OCA2 (Bellono, Escobar et al, 2014), suggesting that SLC45A2 plays an important role in melanogenesis (Montoliu et al, 2014).…”