Kapa express extract kit

The PbPP1c–mCherry line was obtained by single homologous recombination as previously described [14 (link)]. Similarly, a C-terminal mCherry-tagged PbI2 was generated by single homologous recombination of a 1436 pb region of PbI2 without the stop codon (Pr3–Pr2, Supplementary Materials Table S3 and Figure S1A) inserted into the pL1886 vector and NdeI-linearized before transfection into the PbGFP ANKA strain.
Genotyping was performed on total DNA from parental and transfected parasites extracted from schizont pellets using the KAPA Express Extract Kit (KAPA BioSystem Inc, Wilmington, MA, USA). Transfected parasites were genotyped by diagnostic PCR using primers indicated in the Supplementary Materials Figure S1A and Table S3: Pr1–Pr2 for wild locus detection and Pr1–Pr4 for 3’ integration.

pT3TS-zCas9 and T7-gRNA plasmids were from Chen lab^{52 (link)} through Addgene. Following the published protocol^{52 (link)}, nls-zCas9-nls mRNA was synthesized with an mMESSAGE mMACHINE T3 kit (ThermoFisher, AM1348) and recovered with lithium chloride precipitation. lpl gRNA was generated using a MEGAshortscript T7 kit (ThermoFisher, AM1354) and purified using a mirVana miRNA isolation kit (ThermoFisher, AM1560). The zebrafish lpl genomic target sequence was 5′-ggctgaaattgattatccttGGG-3′, in which the first 20 nt was the gRNA template and the last 3 nt was protospacer adjacent motif (PAM) required for CRISPR/Cas9 function. 30 pg lpl gRNA and 150 pg nls-zCas9-nls mRNA were injected into 1–2 cells stage embryos. Genomic DNA (gDNA) was extracted from whole embryos or from adult tail tissue using a KAPA Express Extract Kit (KAPA Biosystems, Cat. KR0383). The gDNA fragment containing the target site was amplified using KOD DNA polymerase (EMD Millipore, Cat. 71086) and digested with T7 endonuclease (NEB, Cat. M0302). Primers used for PCR amplification of lpl gDNA fragment were 5′-aacatcagcctcctacacaa-3′ and 5′-tcactcgtttctcatgcgaa-3′.

To replace the PbRCC-PIP gene by double homologous recombination, a pBS-DHFR vector was used (Guttery et al., 2012 (link)). Fragments corresponding to the 5′ upstream and 3′ downstream sequence of the PbRCC-PIP gene (PBANKA_0820800) were amplified using primers p56-p57 and p58-p59, respectively, and inserted in the pBS-DHFR vector. The construct was linearized before transfection as described (Guttery et al., 2012 (link)).
For HA-tagging of PbPP1, promoter and coding regions of PBANKA_1028300, obtained using primers p38-p39 and p40-p41, respectively, were cloned into the p-TRAD4Ty-TetO7-HA-hDHFR plasmid (Pino et al., 2012 (link)). Before transfection, the sequence was linearized using BglII.
P. berghei ANKA transfections were performed by electroporation of schizont stages according to Janse et al. (2006) (link), using 10 μg of linearized constructs for each transfection. The schizont stages were obtained from 6 week-old infected Fisher rats (Charles River) and separated on a 55% Nycodenz gradient after 16 h culture at 37°C in RPMI1640 culture medium supplemented with 0.4% AlbuMAX^TM II Lipid-Rich BSA (Life Technologies).
Genotyping was performed in parallel on total DNA from parental and transfected parasites extracted from schizont pellets using the KAPA Express Extract Kit (KAPABioSystem). Primers used for genotyping are indicated in Supplementary Figures 2, 4 and Table 1.

All adults were sexed based on apparent sexually dimorphic features, specifically the presence/absence of androconia (the male-specific pheromone organ). Because larvae do not display any obvious sexual dimorphism, we extracted DNA from individuals using the KAPA Express Extract Kit (KAPA Biosystems; Wilmington, MA, USA) and amplified a female-specific W-chromosome microsatellite ([115 (link)]; Genbank accession no.: AY785080) using the primers from [116 (link)]. PCR products were then visualized by gel electrophoresis, and females were identified by the presence of a band at ~ 185 bp, while males lacked this band (Additional file 1: Fig. S9).

Genomic DNA extraction from mouse tissues was performed with KAPA Express Extract Kit (Kapa Biosystems) following the manufacturer’s instruction. PCR reactions were performed with the same kit following KAPA2G Robust PCR Protocol (Kapa Biosystems).
The following primers were used:

Mutations were derived from Chinese MLC patients with GLIALCAM mutations c.274C>T, p.Arg92Trp, c.203A>T(p.Lys68Met), and c.395C>A (p.Thr132Asn). Adult Glialcam^Arg92Trp/+, Glialcam^Lys68Met/+, and Glialcam^Thr132Asn/+ point mutation mice were obtained from the Beijing Vitalstar Biotechnology Co., Ltd. using a CRISPR/Cas9-based knock-in strategy in C57Bl/6N mice (Fig. S2A). Then, we crossbred heterozygous mutant mice between Glialcam^Lys68Met/+ and Glialcam^Thr132Asn/+ mice to obtain the compound heterozygous mouse Glialcam^{Lys68Met/Thr132Asn}. The study was approved by the Animals Ethical Committee of Peking University First Hospital, and animal housekeeping and experiments were in compliance with the guidelines of the committee. All mice were housed in an animal care facility (SPF) with a 12-h light–dark schedule and with freely available food and water. The DNA of the tail was extracted using the KAPA Express Extract kit, and genotype identification of these mice was performed by PCR and Sanger sequencing using the following primers: forward primer 5ʹ-AGGGCTTGAAG TTGGAAATGGGCTA-3ʹ; reverse primer 5′-AATTACCACTCAGCCTTTGCGTTGC-3ʹ. Table S1 records the sex and generations of mice used for the experiment.