Poly-T Scoring

How SPACER detects and penalizes poly-T (TTTT) and poly-U (UUUU) runs that act as premature transcription terminators.

Biological Significance

Runs of four or more consecutive thymine (T) or uracil (U) bases have a specific biological effect beyond general homopolymer concerns: they act as transcription termination signals for RNA Polymerase III (Pol III). Since crRNAs for CRISPR diagnostics are typically expressed from U6 or other Pol III promoters, a TTTT run within the spacer sequence causes premature termination of the crRNA transcript, yielding a truncated, non-functional guide.

This is distinct from the general homopolymer penalty — poly-T runs are biologically disruptive regardless of their effect on binding thermodynamics. Even a single TTTT within the spacer can render the guide unusable when expressed from a Pol III promoter.

Detection

SPACER scans each spacer for consecutive T (DNA mode) or U (RNA mode) runs and records the longest run:

text

Spacer: A T G C C T T T T A G G C T T A A C G T
                      ↑↑↑↑
                  poly-T run (4 nt)

Longest poly-T: 4 → penalty applied

For Cas13 guides (RNA targets), SPACER detects poly-U (UUUU) runs with the same logic. If the input is DNA that undergoes in-silico transcription, poly-T in the DNA maps directly to poly-U in the crRNA.

Scoring Function

The poly-T score is more aggressive than the general homopolymer score, reflecting the severity of the termination effect:

Longest T/U Run	Score	Interpretation
0–3 nt	1.0	No poly-T concern — safe for Pol III expression
4 nt	0.25	Strong penalty — likely premature termination
5 nt	0.1	Very high risk — truncated crRNA almost certain
≥6 nt	0.0	Maximum penalty — guide is effectively unusable

Note that the penalty for a 4-nt poly-T run (0.25) is harsher than the general homopolymer penalty for a 4-nt run (0.5). This reflects the fact that poly-T has a specific, severe biological consequence beyond general homopolymer concerns.

Quality Flag

Flag	Condition	Meaning
POLY_T	Longest T/U run ≥ 4 nt	Spacer contains a poly-T/U sequence that may cause premature Pol III termination

Interaction with Homopolymer Scoring

A poly-T run is also a homopolymer, so guides with poly-T runs are penalized by both the poly-T component and the homopolymer component. This double penalty is intentional — it reflects the fact that poly-T has both the general homopolymer risks (synthesis errors, reduced specificity) and the specific termination risk.

Warning

Guides with a POLY_T flag should be treated with caution regardless of their composite score. If you plan to express crRNAs from a Pol III promoter (U6, H1, or tRNA), even a single TTTT run will likely produce a truncated transcript.

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