Optimized CRISPR-Cpf1 system for genome editing in zebrafish

Abstract

The impact of the CRISPR-Cas biotechnological systems has recently broadened the genome editing toolbox available to different model organisms further with the addition of new efficient RNA-guided endonucleases. We have recently optimized CRISPR-Cpf1 (renamed Cas12a) system in zebrafish. We showed that (i) in the absence of Cpf1 protein, crRNAs are unstable and degraded in vivo, and CRISPR-Cpf1 RNP complexes efficiently mutagenize the zebrafish genome; and (ii) temperature modulates Cpf1 activity especially affecting AsCpf1, which experiences a reduced performance below 37 °C. Here, we describe a step-by-step protocol on how to easily design and generate crRNAs in vitro, purify recombinant Cpf1 proteins, and assemble ribonucleoprotein complexes to carry out efficient mutagenesis in zebrafish in a constitutive and temperature-controlled manner. Finally, we explain how to induce Cpf1-mediated homology-directed repair using single-stranded DNA oligonucleotides. In summary, this protocol includes the steps to efficiently modify the zebrafish genome and other ectothermic organisms using the CRISPR-Cpf1 system.

Keywords: Cas12a; Cpf1; HDR; Temperature regulation; Zebrafish.

Fig. 1.. CRISPR-LbCpf1 compared to CRISPR-SpCas9.

A. Schematic illustrating a LbCpf1 crRNA (binding sequence in green, repeat in blue) binding to the genomic target site (black) and the PAM sequence 5’-TTTV (orange). Red triangles indicate predicted cleavage sites. B. Schematic showing a sgRNA (binding sequence in green, tail in blue) binding to the genomic target site (black) and the PAM sequence 5’-NGG (orange). Red triangles indicate predicted cleavage sites. Adapted from [8].

Fig. 2.. Design of crRNAs with CRISPRscan.org.

A. crRNAs targeting coding-sequences of protein-coding genes. To obtain crRNA, input species and gene in (1) and Cpf1 in (2). CRISPRscan returns available crRNAs indicating, from left to right, Locus, Oligo, number of potential off-targets, and targeted isoform(s). crRNAs with least off-targets are returned first. B. User can submit a specific sequence to target by copy pasting in (3). CRISPRscan returns computed crRNAs after selecting species for off-target prediction and Cpf1 enzyme in (4). Genome browser tracks are available in third tab.

Fig. 3.. PCR approach to obtain a DNA product used as template for crRNA in vitro transcription.

A. An oligonucleotide containing the T7 promoter (red) followed by two Guanine and 20 (AsCpf1) or 21 nt (LbCpf1) of the repeat (tail, in blue) for annealing is used in combination with oligonucleotide containing the reverse complement of the repeat and 23 nt of the binding sequence (in green). A final DNA product of 65bp (AsCpf1) or 66bp (LbCpf1) will be obtained in order to in vitro transcribed mature crRNAs. Adapted from [8]. B. PCR approach to obtain an 80bp (AsCpf1) or 81bp (LbCpf1) product used as template for pre-crRNA in vitro transcription similar to that described above, except the oligonucleotide containing the T7 promoter (red) includes the 35 nt (AsCpf1) or 36 nt (LbCpf1) pre-crRNA repeat sequence (tail, in blue).

Fig. 4.. Cpf1 protein purification.

A. Schematic of the different steps required for the production and purification of Cpf1, and the possible interruptions and storage. B. Example of Heparin ion exchange chromatography purification, where Cpf1 is eluted with a gradient of salt KCl (in green). C. Example of size-exclusion chromatography purification of Cpf1. D. Validation of Cpf1 purification by SDS-PAGE electrophoresis.

Fig. 5.. LbCpf1-mediated homology-directed repair.

A. Schematic illustrating LbCpf1 protein, crRNA and RNP complex interaction with the DNA (TTTV corresponds to the PAM sequence). crRNA annealing occurs on the PAM-non-containing strand (target, blue), and PAM sequences are in the non-target strand (red). B. Asymmetric ssDNA donor must be complementary to the target strand and must have homology arms with different lengths. The PAM-proximal arm (arm that contains the PAM) should be 50 or 71 nt, and the PAM-distal arm should be 36 nt (arm without PAM). PAM sequence in the ssDNA donor was modified (TgaV) to prevent new editing post-HDR event. C-E. Editing strategies. Schematic representation of genomic DNA (red, non-target strand and blue, target strand). RNP complex with crRNA annealing is shown in target strand. ssDNA repair template is depicted below with its respective homology arms, proximal (left) and distal (right) (dashed lines). Final gene product after HDR-mediated integration is shown in gray. Inserts are in blue with gray boxes. Yellow bars represent the Cpf1 staggered DSB. For details, see the corresponding text.