Spacer2PAM: A computational framework to guide experimental determination of functional CRISPR-Cas system PAM sequences

Abstract

RNA-guided nucleases from CRISPR-Cas systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR-Cas systems in many prokaryotes are attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR-Cas effectors. However, each CRISPR-Cas system recognizes a unique set of protospacer adjacent motifs (PAMs), which requires identification by extensive screening of randomized DNA libraries. This challenge hinders development of endogenous CRISPR-Cas systems, especially those based on multi-protein effectors and in organisms that are slow-growing or have transformation idiosyncrasies. To address this challenge, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to predict and guide experimental determination of functional PAM sequences for any CRISPR-Cas system given its corresponding CRISPR array as input. Spacer2PAM can be used in a ‘Quick’ method to generate a single PAM prediction or in a ‘Comprehensive’ method to inform targeted PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-informed PAM library for the type I-B CRISPR-Cas system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR-Cas systems for industrial biotechnology and synthetic biology.

Automated identification of sequence-tailored Cas9 proteins using massive metagenomic data

“…Overall, 18 out of 21 (85%) PAM predictions generated by our method were correct and the remaining 15% were partial predictions with at least one base correctly identified. Our method exhibited a much higher prediction accuracy compared to Spacer2PAM 7 (Supplementary Fig. 3 ), which outperforms both CRISPRTarget and CASPERpam 7 and was therefore chosen as the gold standard for the comparison.…”

“…Our method exhibited a much higher prediction accuracy compared to Spacer2PAM 7 (Supplementary Fig. 3 ), which outperforms both CRISPRTarget and CASPERpam 7 and was therefore chosen as the gold standard for the comparison.…”

“…Spacer2PAM 7 predictions were generated using unique spacer sequences derived from the 98% identity Cas9 clustering. Spacer2PAM (version 0.0.0.9000) was run with default parameters, except the e-value threshold which was set to 0.01.…”

“…To explore the natural PAM-Cas9 diversity, the pre-determination of the PAM requirements is the crucial step. However, available methods to predict Cas9 PAMs present several limitations (Supplementary Table 1 ) preventing their effective exploitation: CASPERpam 6 has very low accuracy (10% functional predictions 7 ); SPAMALOT 8 does not include any spacer match filtering or consensus processing step and its source code is not publicly available; the method described by Vink et al 9 associates PAM predictions with clusters of CRISPR repeats, making it not suited to generate predictions for single Cas9 proteins; CRISPRTarget 10 is only available as a Web service and cannot be automatically interrogated for large sequence repositories; Spacer2PAM 7 can be applied to predict PAMs of single Cas9 proteins but its low accuracy (45% functional predictions 7 ) still impairs comprehensive analyses.…”

PAMPHLET: A Robust Toolkit for Precise PAM Prediction and Unveiling PAM Consistency in Highly Co-occurrence CRISPR-Cas Systems

“…A number of tools, including CRISPRTarget (Biswas et al, 2013), CASPERpam (Mendoza & Trinh, 2018), and Spacer2PAM (Rybnicky et al, 2022), have been developed to collect spacers from the CRISPR array and search the prokaryotic genome database for protospacer adjacent motifs. Spacer2PAM has demonstrated superior performance in guiding the experimental determination of functional PAMs in many instances; however, the quality and quantity of the input spacers are critical for its accuracy.…”